Molecular Biology - The Scripps Research Institute

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Molecular Biology - The Scripps Research Institute

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Molecular Biology


Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Yun Yung, Graduate Student, and Jerold Chun, M.D., Ph.D., Professor,

Department of Molecular Biology


DEPARTMENT OF

MOLECULAR BIOLOGY

STAFF

Peter E. Wright, Ph.D.*

Professor and Chairman

Cecil H. and Ida M. Green

Investigator in Medical

Research

Ruben Abagyan, Ph.D.

Professor

Carlos F. Barbas III, Ph.D.*

Professor

Janet and W. Keith Kellogg II

Chair, Molecular Biology

Michael N. Boddy, Ph.D.

Assistant Professor

Charles L. Brooks III, Ph.D.

Professor

Monica J. Carson, Ph.D.**

Associate Professor

University of California

Riverside, California

David A. Case, Ph.D.

Professor

Geoffrey Chang, Ph.D.*

Associate Professor

Jerold Chun, M.D., Ph.D.

Professor

Lisa Craig, Ph.D.**

Assistant Professor

Simon Fraser University

Burnaby, British Columbia

Valerie De Crecy Lagard,

Ph.D.**

Assistant Professor

University of Florida

Gainesville, Florida

Luis De Lecea, Ph.D.

Associate Professor

Lluis Ribas De Pouplana,

Ph.D.

Adjunct Assistant Professor

Ashok Deniz, Ph.D.

Assistant Professor

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

H. Jane Dyson, Ph.D.

Professor

John H. Elder, Ph.D.

Professor

Martha J. Fedor, Ph.D.*

Associate Professor

James Arthur Fee, Ph.D.

Professor of Research

Elizabeth D. Getzoff,

Ph.D.****

Professor

David B. Goodin, Ph.D.

Associate Professor

David S. Goodsell Jr., Ph.D.

Associate Professor

Joel M. Gottesfeld, Ph.D.

Professor

Robert Hallewell, D.Phil.

Adjunct Associate Professor

Jennifer Harris, Ph.D.

Assistant Professor of

Biochemistry

Christian A. Hassig, Ph.D.

Adjunct Assistant Professor

Mirko Hennig, Ph.D.

Assistant Professor

John E. Johnson, Ph.D.

Professor

Gerald F. Joyce, M.D.,

Ph.D.*****

Professor

Ehud Keinan, Ph.D.

Adjunct Professor

Richard A. Lerner, M.D.,

Ph.D.*****

President, Scripps Research

Lita Annenberg Hazen Professor

of Immunochemistry

Cecil H. and Ida M. Green

Chair in Chemistry

Scott Lesley, Ph.D.

Assistant Professor of

Biochemistry

Tianwei Lin, Ph.D.

Assistant Professor

Clare McGowan, Ph.D. †

Associate Professor

Duncan E. McRee, Ph.D.

Adjunct Associate Professor

David P. Millar, Ph.D.

Associate Professor

Louis Noodleman, Ph.D.

Associate Professor

Arthur J. Olson, Ph.D.

Professor

James C. Paulson, Ph.D. ††

Professor

Vijay Reddy, Ph.D.

Assistant Professor

Steven I. Reed, Ph.D. †

Professor

Victoria A. Roberts, Ph.D.**

Associate Professor

University of California

San Diego, California

Paul Russell, Ph.D.

Professor

MOLECULAR BIOLOGY 2005 155

Michel Sanner, Ph.D.

Associate Professor

Harold Scheraga, Ph.D.

Adjunct Professor

Paul R. Schimmel, Ph.D.*****

Ernest and Jean Hahn

Professor of Molecular

Biology and Chemistry

Anette Schneemann, Ph.D.

Associate Professor

Subhash C. Sinha, Ph.D.*

Associate Professor

Gary Siuzdak, Ph.D.

Adjunct Associate Professor

Robyn L. Stanfield, Ph.D.

Assistant Professor

James Steven, Ph.D.

Assistant Professor

Raymond C. Stevens, Ph.D. †††

Professor

Charles D. Stout, Ph.D.

Associate Professor

Peiqing Sun, Ph.D.

Assistant Professor

J. Gregor Sutcliffe, Ph.D.

Professor

John A. Tainer, Ph.D.*

Professor

Fujie Tanaka, Ph.D.

Assistant Professor

Elizabeth Anne Thomas, Ph.D.

Assistant Professor

SECTION COVER FOR THE DEPARTMENT OF MOLECULAR BIOLOGY: Toll-like

receptors (TLRs) recognize various pathogen-associated molecules and play an important role in

innate immune responses. The human TLR3 recognizes double-stranded RNA from viruses and initiates

an intracellular signaling pathway through the interaction of TIR domains of TLR3 and the

adaptor molecule TRIF. The proposed dimer of the TLR3 ectodomain is displayed on the membrane

surface with double-stranded RNA from viruses. The crystal structure was determined by Jungwoo

Choe, Ph.D., in the laboratory of Ian A. Wilson, D.Phil.


156 MOLECULAR BIOLOGY 2005

James R. Williamson,

Ph.D.*****

Professor

Associate Dean, Kellogg

School of Science and

Technology

Ian A. Wilson, D.Phil.*

Professor

Curt Wittenberg, Ph.D. †

Professor

Kurt Wüthrich, Ph.D.

Cecil H. and Ida M. Green

Professor of Structural

Biology

Todd O. Yeates, Ph.D.

Adjunct Professor

Qinghai Zhang, Ph.D.

Assistant Professor

Guo Fu Zhong, Ph.D.**

Fudan University

Shanghai, China

SERVICE FACILITIES

Ola Blixt, Ph.D.

Core Manager, Consortium

for Functional Glycomics

John Chung, Ph.D.

Manager, Nuclear Magnetic

Resonance Facilities

Gerard Kroon

Assistant Manager, Nuclear

Magnetic Resonance Facilities

Michael E. Pique

Director, Graphics Development

Nahid Razi, Ph.D.

Assistant Core Manager,

Consortium for Functional

Glycomics

Peter Sobieszcsuk, Ph.D.

Core Manager, Consortium

for Functional Glycomics

SENIOR STAFF SCIENTIST

Wayne A. Fenton, Ph.D.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

STAFF SCIENTISTS

Aymeric Pierre De Parseval,

Ph.D.

Karla Ewalt, Ph.D.**

Princeton University

Princeton, New Jersey

Brian M. Lee, Ph.D.

Maria Martinez-Yamout, Ph.D.

Garrett M. Morris, Ph.D.

Chiaki Nishimura, Ph.D.

Jeffrey Speir, Ph.D.

Manal Swairjo, Ph.D.

Mutsuo Yamaguchi, Ph.D.

Xueyong Zhu, Ph.D.

SENIOR RESEARCH

ASSOCIATES

David Barondeau, Ph.D.

Kirk Beebe, Ph.D.

Ryan Burnett, Ph.D.

Brian Collins, Ph.D.

Adrienne Elizabeth Dubin,

Ph.D.

Maria Alejandra Gamez-

Abascal, Ph.D.

Peter B. Hedlund, M.D., Ph.D.

Ying Chuan Lin, Ph.D.

Rebecca Page, Ph.D.**

Brown University

Providence, Rhode Island

Mikhail Popkov, Ph.D.

Richard R. Rivera, Ph.D.

Lincoln Scott, Ph.D.

Koji Tamura, Ph.D.

Liang Tang, Ph.D.**

Burnham Institute

La Jolla, California

Ellie Tzima, Ph.D.**

University of North Carolina

Chapel Hill, North Carolina

Xiang-Lei Yang, Ph.D.

Dirk M. Zajonc, Ph.D.

RESEARCH ASSOCIATES

Sunny Abraham, Ph.D.

Fabio Agnelli, Ph.D.

Moballigh Ahmad, Ph.D.

Alexander Ivanov Alexandrov,

Ph.D.

Marcius Da Silva Almeida,

Ph.D.

Beatriz Gonzalez Alonso, Ph.D.

David Alvarez-Carbonell, Ph.D.

Jianghong An, Ph.D.**

British Columbia Cancer

Agency

Vancouver, British Columbia

Yu An, Ph.D.

Crystal Stacy Anglen, Ph.D.**

Neurome, Inc.

La Jolla, California

Brigitte Anliker, Ph.D.

Roger Armen, Ph.D.

Joseph W. Arndt, Ph.D.

Mabelle Ashe, Ph.D.

Jamie Mitchell Bacher, Ph.D.

Michael F. Bailey, Ph.D.**

Bio21 Institute

Parkville, Victoria, Australia

Manidipa Banerjee, Ph.D.

Christopher Baskerville, Ph.D.

Lipika Basummalick, Ph.D.

Konstantinos Beis, Ph.D.

Per Bengston, Ph.D.

Svitlana Berezhna, Ph.D.

William Henry Bisson, Ph.D.

Pilar Blancafort, Ph.D.**

University of North Carolina

Chapel Hill, North Carolina

David Boehr, Ph.D.

David Bostick, Ph.D.

Ronald M. Brudler, Ph.D.

Lintao Bu, Ph.D.

Rosa Maria Cardoso, Ph.D.

Justin E. Carlson, Ph.D.

Andrew Barry Carmel, Ph.D.

Qing Chai, M.D., Ph.D.

Brian Chapados, Ph.D.

Eli Chapman, Ph.D.

Anju Chatterji, Ph.D.

Anton Vladislavovich

Cheltsov, Ph.D.

Jianhan Chen, Ph.D.

Yen-Ju Chen, Ph.D.

Zhiyong Chen, Ph.D.

Jaeyoung Cho, Ph.D.**

Hallym University

Kangwon, South Korea

Jungwoo Choe, Ph.D.

Chung Jen Chou, Ph.D.

Li-Chiou Chuang, Ph.D.

Jean-Pierre Clamme, Ph.D.


Linda Maria Columbus, Ph.D.

Adam Corper, Ph.D.

Qizhi Cui, Ph.D.

Carla P. Da Costa, Ph.D.

Douglas Daniels, Ph.D.**

Yale University

New Haven, Connecticut

Sanjib Das, Ph.D.

Paramita Dasgupta, Ph.D.**

Mayo Clinic

Rochester, Minnesota

Robert De Bruin, Ph.D.

Roberto N. De Guzman,

Ph.D.**

University of Kansas

Lawrence, Kansas

Sohela De Rozieres, Ph.D.

Qingdong Deng, Ph.D.

Paula Desplats, Ph.D.

Buchi Ramachary

Dhevalapally, Ph.D.**

University of Hyderabad

Hyderabad, India

Claire Louise Dovey, Ph.D.

Zhanna Druzina, Ph.D.

Li-Lin Du, Ph.D.

Theresia Dunzendorfer-Matt,

Ph.D.**

Leopold Franzens Universität

Innsbruck, Austria

Scott Eberhardy, Ph.D.

Marc-Olivier Ebert, Ph.D.**

Leopold Franzens Universität

Innsbruck, Austria

Stephen Edgcomb, Ph.D.

Susanna V. Ekholm-Reed,

Ph.D.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Reza Mobini Farahani, Ph.D.**

Sahlgrenska University

Hospital

Göteborg, Sweden

Daniel Felitsky, Ph.D.

Allan Chris Merrera Ferreon,

Ph.D.

Josephine Chu Ferreon, Ph.D.

Pierre Henri Gaillard, Ph.D.

Hui Gao, Ph.D.

Elsa D. Garcin, Ph.D.

Shannon E. Gardell, Ph.D.

Edith Caroline Glazer, Ph.D.

Bettina Groschel, Ph.D.

Björn Grünenfelder, Ph.D.**

Novartis Institutes for

BioMedical Research

Cambridge, Massachusetts

Fang Guo, Ph.D.

Gye Won Han, Ph.D.

Hongna Han, Ph.D.**

American BioScience, Inc.

Santa Monica, California

Shoufa Han, Ph.D.

Wenge Han, Ph.D.

Jason W. Harger, Ph.D.

Brian Henriksen, Ph.D.**

Eurogentec North America, Inc.

San Diego, California

David M. Herman, Ph.D.

Deron Herr, Ph.D.

Kenichi Hitomi, Ph.D.

Reto Horst, Ph.D.

Yunfeng Hu, Ph.D.

Joy Huffman, Ph.D.**

McKinsey & Company

Los Angeles, California

Laura M. Hunsicker, Ph.D.**

Trinity University

San Antonio, Texas

Kwan Hoon Hyun, Ph.D.

Wonpil Im, Ph.D.

Tasneem Islam, Ph.D.**

University of Melbourne

Melbourne, Australia

Shuichiro Ito, Ph.D.**

Sankyo Co., Ltd.

Tokyo, Japan

Kai Jenssen, Ph.D.

Glenn C. Johns, Ph.D.

Eric C. Johnson, Ph.D.

Margaret Alice Johnson, Ph.D.

Hamid Reza Kalhor, Ph.D. ††††

Christian Kannemeier, Ph.D.

Mili Kapoor, Ph.D.

Andrey Aleksandrovich

Karyakin, Ph.D.

Yang Khandogin, Ph.D.

Ilja V. Khavrutskii, Ph.D.

Reza Khayat, Ph.D.

Dae Hee Kim, Ph.D.

Min Ju Kim, Ph.D.**

Genomics Institute of the

Novartis Research Foundation

San Diego, California

Eda Koculi, Ph.D.

Milka Kostic, Ph.D.

Julio Kovacs, Ph.D.

Irina Kufareva, Ph.D.

MOLECULAR BIOLOGY 2005 157

Shantanu Kumar, Ph.D.

Iaroslav Kuzmin, Ph.D. ††††

Hugo Alfredo Lago-Zarrilli,

Ph.D. ††††

Bianca Lam, Ph.D.

Polo Chun Hung Lam, Ph.D.

Emma Langley, Ph.D.

Jason Lanman, Ph.D.

Jonathan C. Lansing, Ph.D.**

Momenta Pharmaceuticals

Cambridge, Massachusetts

Chang-Wook Lee, Ph.D.

Chul Won Lee, Ph.D.

Jinhyuk Lee, Ph.D.

June Hyung Lee, Ph.D.

Kelly Lee, Ph.D.

Katrina Lehmann, Ph.D. ††††

Chenglong Li, Ph.D.

Vasco Liberal, Ph.D.

William M. Lindstrom, Ph.D.

Hui-Yue Christine Lo, Ph.D.

Kunheng Luo, Ph.D.

John Gately Luz, Ph.D.**

Harvard University

Boston, Massachusetts

Che Ma, Ph.D.**

Academia Sinica

Taipei, Taiwan

Ann MacLaren, Ph.D.

Laurent Magnenat, Ph.D.**

Serono Pharmaceutical

Research Institute SA

Geneva, Switzerland

Darly Joseph Manayani, Ph.D.


158 MOLECULAR BIOLOGY 2005

Jeff Mandell, Ph.D.

Maria Victoria Martin-

Sanchez, Ph.D.

Tsutomu Matsui, Ph.D.

Daniel McElheny, Ph.D.**

University of Chicago

Chicago, Illinois

Benoit Melchior, Ph.D.**

University of California

Riverside, California

David Metzgar, Ph.D.**

Naval Health Research Center

San Diego, California

Jonathan Mikolosko, Ph.D.

Susumu Mitsumori, Ph.D.

Heiko Michael Moeller,

Ph.D.**

Universität Konstanz

Konstanz, Germany

Seongho Moon, Ph.D.

Bettina Moser, Ph.D.**

University of Illinois at Chicago

Chicago, Illinois

Samrat Mukhopadhyay, Ph.D.

Christopher Myers, Ph.D.**

Naval Health Research Center

San Diego, California

Sreenivasa Chowdari Naidu,

Ph.D.**

MediVas, L.L.C.

San Diego, California

Toru M. Nakamura, Ph.D.**

University of Illinois at Chicago

Chicago, Illinois

Sujatha Narayan, Ph.D.

Hung Nguyen, Ph.D.

Tadateru Nishikawa, Ph.D.

Eishi Noguchi, Ph.D.**

Drexel University

Philadelphia, Pennsylvania

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Wataru Nomura, Ph.D.

Brian Nordin, Ph.D.**

ActivX Biosciences, Inc.

La Jolla, California

Karin E. Norgard-Sumnicht,

Ph.D.**

San Diego State University

San Diego, California

Brian V. Norledge, Ph.D.

Michael Oberhuber, Ph.D.**

Leopold Franzens Universität

Innsbruck, Austria

Wendy Fernandez Ochoa,

Ph.D.

Amy Odegard, Ph.D.

Yoshiaki Zenmei Ohkubo,

Ph.D.**

Rutgers University

Piscataway, New Jersey

Brian L. Olson, Ph.D.

Brian Paegel, Ph.D.

Covadonga Paneda, Ph.D.**

Molecular and Integrative

Neurosciences Department,

Scripps Research

Sandeep Patel, Ph.D.

Natasha Paul, Ph.D.**

Stratagene, Inc.

La Jolla, California

Stephanie Pebernard, Ph.D.

Suzanne Peterson, Ph.D.**

University of California

San Diego, California

Wolfgang Stefan Peti, Ph.D.**

Brown University

Providence, Rhode Island

Goran Pljevaljcic, Ph.D.

Corinne Chantal Ploix, Ph.D.**

Novartis International AG

Basel, Switzerland

Stephanie Pond, Ph.D.

Owen Pornillos, Ph.D.

Daniel Joseph Price, Ph.D.

Plachikkat Krishnan Radha,

Ph.D. ††††

Grazia Daniela Raffa, Ph.D.

John Reader, Ph.D.**

University of North Carolina

Chapel Hill, North Carolina

Stevens Kastrup Rehen,

Ph.D.**

Universidade Federal do Rio

de Janeiro

Rio de Janeiro, Brazil

Jean-Baptiste Reiser, Ph.D.**

European Synchrotron

Radiation Facility

Grenoble, France

Miguel A. Rodriguez-

Gabriel, Ph.D.**

Universidad Complutense de

Madrid

Madrid, Spain

Stanislav Rudyak, Ph.D.

Sean Ryder, Ph.D.

Sanjay Adrian Saldanha, Ph.D.

Sanjita Sasmal, Ph.D. ††††

Mika Aoyagi Scharber, Ph.D.**

Burnham Institute

La Jolla, California

Jennifer S. Scorah, Ph.D.

Pedro Serrano-Navarro, Ph.D.

Craig McLean Shepherd, Ph.D.

William Shih, Ph.D**

Dana Farber Cancer Institute

Boston, Massachusetts

David S. Shin, Ph.D.

Develeena Shivakumar, Ph.D.

Holly Heaslet Soutter, Ph.D.

Natalie Spielewoy, Ph.D.**

Weatherall Institute of

Molecular Medicine

Oxford, England

Greg Springsteen, Ph.D.

Deborah J. Stauber, Ph.D.**

Novartis Institutes for

BioMedical Research

Cambridge, Massachusetts

Derek Steiner, Ph.D.**

Johnson & Johnson

San Diego, California

Gudrun Stengel, Ph.D.

Daniel Stoffler, Ph.D.**

Universität Basel

Basel, Switzerland

Kenji Sugase, Ph.D.

Vidyasankar Sundaresan,

Ph.D.**

GE Infrastructure

Trevose, Pennsylvania

Magnus Sundstrom, Ph.D.

Jeff Suri, Ph.D.**

GluMetrics, Inc.

Long Beach, California

Blair R. Szymczyna, Ph.D.

Florence Muriel Tama, Ph.D.

Jinghua Tang, Ph.D.**

University of California

San Diego, California

Nardos Tassew, Ph.D.

Hiroaki Tateno, Ph.D.

Michela Taufer, Ph.D.**

University of Texas

El Paso, Texas

Ewan Richardson Taylor, Ph.D.

Donato Tedesco, Ph.D.**

Berlex Biosciences

Richmond, California


Hua Tian, Ph.D.

Rhonda Torres, Ph.D.**

Merck & Co.

Rahway, New jersey

Megan Wright Trevathan,

Ph.D.**

Harvard Medical School

Boston, Massachusetts

Ulrich Ignaz Tschulena, Ph.D.

Julie L. Tubbs, Ph.D.

Naoto Utsumi, Ph.D.

Frank van Drogen, Ph.D.

Philip Arno Venter, Ph.D.

Petra Verdino, Ph.D.

Stefan Vetter, Ph.D.**

Florida Atlantic University

Boca Raton, Florida

William Frederick Waas,

Ph.D.

Shun-ichi Wada, Ph.D.

Ross Walker, Ph.D.

Robert Scott Williams, Ph.D.

Raphaelle Winsky-

Sommerer, Ph.D.**

Universität Zürich

Zürich, Switzerland

Eric L. Wise, Ph.D.

Jonathan Wojciak, Ph.D.

Dennis Wolan, Ph.D.**

Sunesis Pharmaceuticals,

Inc.

South San Francisco,

California

Hyung Sik Won, Ph.D.**

Konkuk University

Chungju, Korea

Timothy I. Wood, Ph.D.

Eugene Wu, Ph.D.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Lan Xu, Ph.D.

Yoshiki Yamada, Ph.D.

Atsushi Yamagata, Ph.D.

Qi Yan, Ph.D.

Yong Yao, Ph.D.

Xiaoqin Ye, M.D., Ph.D.

Yongjun Ye, Ph.D.

Yong Yin, Ph.D.

Veronica Yu, Ph.D.

Yuan Yuan, Ph.D.

Markus Zeeb, Ph.D.

Ying Zeng, Ph.D.

Haile Zhang, Ph.D.

Yong Zhao, Ph.D.

Peizhi Zhu, Ph.D.

SCIENTIFIC ASSOCIATES

Enrique Abola, Ph.D.

Andrew S. Arvai, M.S.

Eric Birgbauer, Ph.D.

Ognian V. Bohorov, Ph.D.

Dennis Carlton, B.S.

Ellen Yu-Lin Tsai Chien,

Ph.D.

Xiaoping Dai, Ph.D.

Liliane Dickinson, Ph.D. ††††

Michael Allen Hanson,

Ph.D.

Diane Marie Kubitz, B.A.

Marcy A. Kingsbury, Ph.D.

Rolf Mueller, Ph.D.

Padmaja Natarajan, Ph.D.

Marianne Patch, Ph.D.

Gabriela Perez-Alvarado,

Ph.D.

Nicholas Preece, Ph.D.

Lin Wang, Ph.D.

VISITING

INVESTIGATORS

Stephen J. Benkovic, Ph.D.

Pennsylvania State University

University Park, Pennsylvania

Astrid Graslund, Ph.D.

Stockholm University

Stockholm, Sweden

Arne Holmgren, M.D., Ph.D.

Karolinska Institutet

Stockholm, Sweden

Barry Honig, Ph.D.

Columbia University

New York, New York

Arthur Horwich, M.D.

Yale University

New Haven, Connecticut

Tai-huang Huang, Ph.D.

Academica Sinica

Taipei, Taiwan

Robert D. Rosenstein, Ph.D.

Lawrence Berkeley National

Laboratory

Berkeley, California

MOLECULAR BIOLOGY 2005 159

* Joint appointment in The Skaggs

Institute for Chemical Biology

** Appointment completed; new

location shown

*** Joint appointment in the

Molecular and Integrative

Neurosciences Department

**** Joint appointments in the

Department of Immunology and

The Skaggs Institute for

Chemical Biology

***** Joint appointments in the

Department of Chemistry and

The Skaggs Institute for

Chemical Biology

† Joint appointment in the

Department of Cell Biology

†† Joint appointment in the

Department of Molecular and

Experimental Medicine

††† Joint appointment in the

Department of Chemistry

†††† Appointment completed


160 MOLECULAR BIOLOGY 2005

Chairman’s Overview

Research in the Department of Molecular Biology

encompasses a broad range of disciplines, extending

from structural and computational biology at

one extreme to molecular genetics at the other. During

the past year, our scientists continued to make rapid

progress toward understanding the fundamental molecular

events that underlie the processes of life. Major

advances have been made in elucidating the structural

biology of signal transduction and viral assembly, in

understanding mechanisms of viral infectivity, in determining

the structure of membrane proteins, in understanding

the molecular basis of nucleic acid recognition

and DNA repair, and in determining the mechanism of

ribosome assembly. Progress was made in elucidating

the molecular events involved in regulation of the cell

cycle, in tumor development, in induction of sleep, in

the molecular origins of neuronal development and of

CNS disorders, in the regulation of transcription, and in

the decoding of genetic information in translation. Finally,

new advances were made in the design of novel low

molecular weight compounds that can specifically regulate

genes and in the area of biomolecular engineering,

building novel functions into viruses, antibodies, zinc

finger proteins, RNA, and DNA. Progress in these and

other areas is described in detail on the following pages,

and only a few highlights are mentioned here.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Peter E. Wright, Ph.D.

Structural biology continues to be a major activity

in the department, and many new x-ray and nuclear

magnetic resonance structures of major biomedical

importance were completed during the past year. Among

the highlights was the determination, in Ian Wilson’s laboratory,

of the first structure of a human Toll-like receptor,

a protein that plays a key role in the innate immune

system as a sensor of molecules associated with the cell

wall and genetic material of pathogenic bacteria. Dr. Wilson

and his coworkers also reported structures of the

protein CD1a, another key receptor in the innate immune

response, and of an antibody that neutralizes most strains

of HIV. Other advances came in the area of membrane

protein crystallography: Geoffrey Chang and colleagues

determined the structures of 2 proteins (MsbA and EmrE)

involved in drug transport and the development of drug

resistance in bacteria and cancer cells, and David Stout

and James Fee determined the structure of a cytochrome

ba 3 oxidase. Finally, the Joint Center for Structural

Genomics, directed by Ian Wilson, was selected by the

National Institutes of Health as 1 of 4 large-scale centers

for high-throughput determination of protein structures.

Several research groups are working in areas directly

related to drug discovery and protein therapeutics. Joel

Gottesfeld and colleagues have developed a small DNAbinding

molecule that turns off the gene for histone H4

and blocks replication in a wide variety of cancer cells.

The compound is active in vivo and blocks the growth

of tumors in mice. Research in the laboratory of Carlos

Barbas is directed toward genetic reprogramming of

tumor cells via engineered zinc finger transcription factors.

These artificial transcription factors are powerful

tools for determining the function of genes in tumor

growth and progression and have potential applications

in cancer therapy. John Elder and colleagues are studying

development of resistance to drugs that target the

HIV protease. A complementary approach to the same

problem is being taken by Arthur Olson and researchers

in his laboratory in their FightAIDS@Home program.

This program is a large-scale computational effort in

which a grid of personal computers distributed around

the world is used to design effective therapeutic agents

that target the HIV protease. Raymond Stevens and

coworkers have engineered a phenylalanine ammonia

lyase enzyme as a potential injectable therapeutic agent

for treating phenylketonuria. Finally, Paul Schimmel and

colleagues have identified a naturally occurring fragment

of tryptophanyl-tRNA synthetase that is highly potent in

arresting angiogenesis and is being introduced in a clinical

setting for treatment of macular degeneration.


Many of the research groups in the department are

applying the tools of molecular genetics to understand

the molecular basis of human disease. Jerold Chun and

his colleagues recently established a relationship between

lysophospholipid signaling and neuropathic pain. In addition,

they made the surprising discovery that lysophosphatidic

acid receptors play an important role in embryonic

implantation and thereby influence female fertility.

Research in the laboratory of Luis de Lecea has indicated

that a newly discovered neuropeptide, neuropeptide S,

plays a functional role in modulation of sleep and suppression

of anxiety. Work in the laboratory of James

Paulson has led to the development of novel microarray

technology for profiling glycoproteins, a technology that

could eventually be developed into a powerful diagnostic

screen for various infections and diseases.

On the more fundamental side, major advances have

been made in understanding mechanisms of protein and

RNA folding, both in vitro and in a cellular environment.

Research in the laboratory of Martha Fedor has resulted

in new insights into mechanisms by which RNA folds

into its specific functional structures and has provided

evidence that RNA chaperones mediate folding pathways

in the cell. Work by James Williamson and colleagues

has led to a detailed map of the assembly landscape of

the 30S ribosome, providing new understanding of the

mechanism by which assembly proceeds through a succession

of RNA conformational changes and protein

binding events. Arthur Horwich and coworkers have

made major progress in elucidating the mechanism by

which the chaperone ClpA mediates unfolding and translocation

of proteins.

Molecular biology remains a field of enormous opportunity

and excitement. The scientists in the department

are taking full advantage of powerful new technologies

to advance our understanding of fundamental biological

processes at the molecular level. Their discoveries will

ultimately be translated into new advances in biotechnology

and in medicine.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 161


162 MOLECULAR BIOLOGY 2005

INVESTIGATORS’ REPORTS

Structural Biology of

Immune Recognition,

Molecular Assemblies,

and Anticancer Targets

I.A. Wilson, R.L. Stanfield, J. Stevens, X. Zhu, Y. An,

K. Beis, T.A. Bowden, D.A. Calarese, R.M.F. Cardoso,

P.J. Carney, J.-W. Choe, A.L. Corper, M.D.M. Crispin,

T.A. Cross, X. Dai, W.L. Densley, E.W. Debler, M.-A. Elsliger,

S. Ferguson, G.W. Han, P.A. Horton, S. Ito, M.J. Jimenez-

Dalmaroni, M.S. Kelker, J.G. Luz, J.B. Reiser,

E.B. Shillington, D.A. Shore, D.J. Stauber, R.S. Stefanko,

J.A. Vanhnasy, P. Verdino, E. Wise, D.W. Wolan, L. Xu,

M. Yu, D.M. Zajonc, Y. Zhang

Our main research focus is concerned with macromolecules

and molecular complexes related to

the innate and adaptive immune responses, viral

pathogenesis, protein trafficking, purine biosynthesis, and

reproductive biology. We use x-ray crystallography to

determine atomic structures of key proteins in these systems

in order to interpret functional data to probe mechanisms

and modes of interaction and to aid in the design

of therapeutic agents as potential drugs or vaccines.

THE INNATE IMMUNE SYSTEM

Toll-like receptors (TLRs) are important mammalian

glycoproteins involved in innate immunity that recognize

conserved structures in pathogens called pattern recognition

motifs. We recently determined the 2.1-Å crystal

structure of the extracellular domain of human TLR3,

which is activated by double-stranded viral RNA. TLR3

forms a large horseshoelike structure with an outer diameter

of 80 Å. Key features include a hydrophobic core

formed by the conserved leucine-rich repeats and a

continuous β-sheet that spans 270° of arc. We are also

investigating other TLRs and their ligands to understand

how microorganisms are initially sensed by the innate

immune system. Our goal is to use the data to design

novel selective agonists and antagonists of TLR signaling

pathways. This research is being done in collaboration

with R.J. Ulevitch and B. Beutler, Department

of Immunology.

Another family of pattern recognition molecules called

peptidoglycan recognition proteins (PGRPs) interacts

with peptidoglycans. We have determined the crystal

structure of the “recognition” PGRP-SA at 1.56 Å. Com-

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

parison of PGRP-SA with a “catalytic” PGRP-LB indicates

overall structural conservation and a hydrophilic

groove that most likely corresponds to the peptidoglycan

core binding site.

Approximately 22,500 intensive care patients across

the United States die of septic shock syndrome every

year. Recently, researchers found that a newly discovered

receptor termed triggering receptor expressed on myeloid

cells 1 (TREM-1) mediates septic shock. We determined

structures of human and mouse TREM-1 immunoglobulin-type

domains to 1.47 Å and 1.76 Å, respectively.

These structural results provided insights into the nature

of ligand recognition by the TREM family in innate immunity.

The studies on TREMs and PGRPs are being done in

collaboration with L. Teyton, Department of Immunology.

CLASSICAL AND NONCLASSICAL MHC AND T-CELL

RECEPTOR SIGNALING

In cellular immunity, T-cell receptors (TCRs) sense

invading pathogens by recognizing pathogen-derived peptide

fragments presented by MHC molecules. The TCRs

then act in concert with CD8 and CD3, which assist in

transducing the antigen recognition signal. Aberrant signaling

can result in numerous disease states. The αβ TCR

coreceptor CD8 is an essential factor in the TCR-mediated

activation of cytotoxic T lymphocytes. We are doing structural

studies of the CD8αβ and the CD8αα isoforms and

of other constituents of the TCR signaling complex.

The CD1 family of nonclassical MHC molecules presents

lipid antigens to CD1-restricted TCRs. Our recent

crystal structure of mouse CD1d at 2.2 Å in complex

with the exceptionally potent short-chain sphingolipid

α-galactosyl ceramide (Fig. 1) reveals a precise hydro-

Fig. 1. The short-chain sphingolipid α-galactosyl ceramide bound

to mouse CD1d. This sphingolipid is a strong agonist of natural killer

T cells. Both alkyl chains of the ligand are buried deep inside the

binding groove, whereas the galactose headgroup is optimally positioned

on top of the binding groove to directly interact with the TCR.


gen-bonding network that positions the galactose moiety.

Other CD1 structures determined include those of

CD1a with a bound sulfatide and with a lipopeptide

that have revealed how dual- and single-chain lipids

interact with the same CD1 molecule. Collaborators in

this research include D.B. Moody and M.B. Brenner,

Harvard Medical School, Boston, Massachusetts; C.-H.

Wong, Department of Chemistry; L. Teyton, Department

of Immunology; M. Kronenberg, La Jolla Institute for

Allergy and Immunology, San Diego, California; V. Kumar,

Torrey Pines Institute for Molecular Studies, San Diego,

California; and Wayne Severn, AgResearch, Upper Hut,

New Zealand.

1918 INFLUENZA VIRUS

Flu is a contagious respiratory disease caused by

influenza viruses. Of all the known pandemics in the

history of humans, the 1918 influenza outbreak was

the most destructive; according to estimates, 40 million

persons died. As a member of the “flu consortium”

funded by the National Institutes of Health, we are

working toward a molecular understanding of why this

particular influenza virus was so pathogenic and how

it managed to evade the immune system so effectively.

We have determined the structure of the hemagglutinin

of the 1918 virus, and now we are investigating

the other viral proteins. We recently analyzed the receptor

specificity of the 1918 hemagglutinin by comparing

its binding to a panel of carbohydrates with the binding

of more modern human and avian viruses (Fig. 2). For

these studies, we are using novel glycan array technology

developed by O. Blixt and J. Paulson, Consortium

for Functional Glycomics, La Jolla, California.

HIV TYPE 1 NEUTRALIZING ANTIBODIES

A vaccine effective against the HIV type 1 must

elicit antibodies that neutralize all circulating strains of

the virus. However, antibodies with such properties are

extremely rare; to date, only a handful have been isolated.

Crystal structures for 4 of these rare, potent,

broadly neutralizing antibodies (b12, 2G12, 4E10,

447-52D) in complex with their viral antigens have

revealed the structural basis for the effectiveness of the

antibodies (Fig. 3). Our goal is to design compounds on

the basis of this structural information (retrovaccinology)

for testing as potential vaccines. The research on HIV

is being done in collaboration with D. Burton, Department

of Immunology; P. Dawson, Department of Cell

Biology; C.-H. Wong, Department of Chemistry; S. Danishefsky,

Sloan-Kettering Institute, New York, New York;

J.K. Scott, Simon Fraser University, Burnaby, British

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The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 163

Fig. 2. Results for carbohydrate array binding of the 2 natural

hemagglutinins from the influenza virus that circulated during the

1918 pandemic. Human-adapted viruses preferentially bind to receptors

with a terminal sialic acid linked by an α2,6 linkage to a vicinal

galactose, whereas avian-adapted viruses recognize an α2,3 linkage.

Glycan array results are shown for 18SC (A/South Carolina/1/18; A),

and 18NY (A/New York/1/18; B). These 2 hemagglutinins differ by

a single point mutation that is sufficient to alter the carbohydrate specificity

from exclusively α2,6 to mixed α2,6/α2,3. AGP indicates α1- acid glycoprotein.

Fig. 3. Antigen binding site of the Fab fragment of 4E10, an

antibody to gp41. 4E10 cross-reacts with more viral isolates (clades)

than any other known HIV type 1 neutralizing antibody. The crystal

structure of Fab 4E10 is shown in complex with a synthetic peptide

that encompasses the highly conserved 4E10 epitope. The peptide

(ball and stick) binds to the surface of Fab 4E10 (solid surface) in

a shallow hydrophobic cavity in a helical conformation. The structure

also suggests that the complementarity-determining region H3

loop of 4E10 may contact the cell membrane, because the loop is

adjacent to the membrane-proximal epitope.


164 MOLECULAR BIOLOGY 2005

Columbia; S. Zolla-Pazner, New York University School

of Medicine, New York, New York; J. Moore, Cornell

University, Ithaca, New York; Repligen Corporation,

Waltham, Massachusetts; H. Katinger, R. Kunert, and

G. Stiegler, University für Bodenkultur, Vienna, Austria;

and R. Wyatt and P. Kwong, Vaccine Research Center,

National Institutes of Health, Bethesda, Maryland.

PRIMITIVE IMMUNOGLOBULINS

Cartilaginous fish are the phylogenetically oldest

living organisms known to have components of the

vertebrate adaptive immune system, such as antibodies,

MHC molecules, and TCRs. Key to their immune

response are heavy-chain, homodimeric immunoglobulins

(“new antigen receptors” or IgNARs) in which the

antigen-recognizing variable domains consist of only a

single immunoglobulin domain. In collaboration with

M. Flajnik, University of Maryland Medical School, Baltimore,

Maryland, we determined the crystal structure for

an IgNAR variable domain in complex with its lysozyme

antigen (Fig. 4). The results revealed that 2 complementarity-determining

regions are sufficient for antigen

recognition. These and ongoing studies will determine

whether the IgNAR variable domains are an evolutionary

precursor to mammalian TCR and antibody

immunoglobulin domains.

CATALYTIC ANTIBODIES

Catalytic antibodies can be generated to carry out

many difficult and novel chemical reactions, including

Fig. 4. Nurse shark IgNAR type I variable domain (tubes) bound

to its lysozyme antigen (solid surface). The IgNAR variable domains

have an unusual antigen-binding site that contains only 2 of the 3

conventional complementarity-determining regions (CDRs), but it still

binds antigen with nanomolar affinity via an interface comparable

in size to conventional antibodies. Two other regions, HV2 and HV4,

are also somatically mutated, suggesting that they may also be

involved in antigen recognition for other IgNAR-antigen complexes.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

reactions not catalyzed by naturally occurring enzymes.

Examples currently under study include several cocainehydrolyzing

antibodies that could act as possible therapeutic

agents to counter cocaine overdose or addiction,

highly efficient but widely acting aldolase antibodies,

and antibodies that carry out proton abstraction from

carbon (Fig. 5). The studies on catalytic antibodies

are being done in collaboration with R.A. Lerner, C.F.

Barbas, K.D. Janda, P.G. Schultz, F. Tanaka, P. Wentworth,

and P. Wirsching, Department of Chemistry;

D.W. Landry, Columbia University, New York, New York;

and D. Hilvert, ETH Zürich, Zürich, Switzerland.

Fig. 5. Antibody-combining site of 34E4 bound to hapten. Catalytic

antibody 34E4 catalyzes the conversion of benzisoxazoles to

salicylonitriles with high rates and multiple turnovers. This reaction

is a widely used model system for studies of proton abstraction from

carbon. The structure of 34E4 in complex with its hapten has revealed

many similarities to biological counterparts that promote proton transfers.

Nevertheless, the reliance of 34E4 on a single catalytic residue

(GluH50 ) probably prevents it from achieving the rates of the

most efficient enzymes. Two of the active-site water molecules are

designated S1 and S21. The 3Fo-2Fc σA-weighted electron density

map around the hapten and key active-site residues is contoured at

1.3 σ. Hydrogen bonds are shown as broken lines. TrpL91 forms a

cation-π interaction with the guanidinium moiety of the hapten.

EVOLUTION OF LIGAND RECOGNITION AND

SPECIFICITY

The antibodies 1E9 and DB3 share a human germline

precursor but recognize different ligands. Residues

in the Diels-Alderase antibody 1E9 active site have

been sequentially mutated by D. Hilvert to change the

specificity of 1E9 to that of the steroid-binding DB3.


Only 2 key residues in 1E9 are required to switch

between the catalytic antibody activity and steroid binding

that is 14,000-fold higher than in the original 1E9

antibody. Crystal structures of these steroid-bound 1E9

mutants show that although 1E9 and DB3 share similar

steroid-binding properties, they surprisingly accomplish

binding and specificity in a structurally distinct manner.

BLUE AND PURPLE FLUORESCENT ANTIBODIES

Antibodies generated against trans-stilbene have

an interesting, unexpected photochemistry when bound

to that hapten. Several of these antibodies bind stilbene

with high affinity, yet have significantly different

spectroscopic properties. Crystal structures have now

been determined to probe the antibodies’ mechanism

of action, and further biophysical and biochemical

studies are being performed in the laboratories of our

collaborators, R.A. Lerner, Department of Molecular

Biology; K.D. Janda and F.E. Romesberg, Department

of Chemistry; and H.G. Gray, California Institute of Technology,

Pasadena, California.

PROTEIN TRAFFICKING

The Rab family GTPases are ubiquitously involved

in regulation of membrane docking and fusion in endocytic

and exocytic pathways. The tethering factor p115

is recruited by Rab1 to vesicles of coat protein complex

II during budding from the endoplasmic reticulum

and subsequently interacts with a set of SNARE proteins

associated with the vesicles to promote targeting

to the Golgi complex. In collaboration with W.E. Balch,

Department of Cell Biology, we determined the crystal

structure of p115 at 2.0 Å and localized the binding

site on p115 for Rab1 by mutational analysis.

ENZYMATIC CANCER TARGETS

The de novo purine biosynthesis pathway is the primary

provider of purine nucleotides, which are converted

to DNA building blocks. This biosynthesis pathway is

a validated target for the development of anticancer

drugs because of heavy dependence on it by fast-growing

cells, such as tumor cells. We have focused on 2

folate-dependent enzymes in the pathway: glycinamide

ribonucleotide transformylase and the bifunctional aminoimidazole

carboxamide ribonucleotide transformylase

inosine monophosphate cyclohydrolase (ATIC, Fig. 6).

Crystal structures of these 2 enzymes in complex with

many different classes of inhibitors have provided a valuable

platform for development of antineoplastic agents.

These investigations are being done in collaboration with

D.L. Boger, Department of Chemistry; A.J. Olson, Department

of Molecular Biology; G.P. Beardsley, Yale Univer-

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The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 165

Fig. 6. The active site of ATIC in complex with a novel nonfolate

inhibitor identified by virtual ligand screening. The inhibitor is depicted

in ball-and-stick representation and is surrounded by 2Fo-Fc electron

density contoured at 1σ.

sity, New Haven, Connecticut; and S.J. Benkovic, Pennsylvania

State University, University Park, Pennsylvania.

GHMP KINASES IN REPRODUCTIVE BIOLOGY

XOL-1 is the primary sex-determining signal from

Caenorhabditis elegans. The crystal structure of XOL-1

revealed that the protein belongs to the GHMP kinase

family of small-molecule kinases, establishing an unanticipated

role for this protein family in differentiation and

development. In collaboration with B.J. Meyer, University

of California, Berkeley, California, we identified

XOL-1 homologs in the genomes of Caenorhabditis

briggsae and Caenorhabditis remanei and are examining

their function by using suppression of gene expression

mediated by RNA interference. Although XOL-1 is

structurally similar to its GHMP kinase neighbors, its

endogenous ligand is unknown. Using the crystal structure

of XOL-1 as a template for virtual screening, we

identified several potential synthetic XOL-1 ligands, and

in collaboration with J.R. Williamson, Department of

Molecular Biology, we confirmed their binding by using

nuclear magnetic resonance.

JOINT CENTER FOR STRUCTURAL GENOMICS

The Joint Center for Structural Genomics is a large

consortium of scientists from Scripps Research, the

Stanford Synchrotron Radiation Laboratory, the University

of California, San Diego, the Burnham Institute, and

the Genomics Institute of the Novartis Research Foundation.

The center is funded by the Protein Structure


166 MOLECULAR BIOLOGY 2005

Initiative of the National Institute of General Medical

Sciences. Its purpose is the high-throughput structure

determination of the complete proteomes of a procaryote,

Thermotoga maritima, and a eukaryote, the mouse.

To date, members of the consortium have pioneered

the development of many novel high-throughput methods,

constructed a high-throughput pipeline, and determined

more than 200 nonredundant structures, including

100 in the past year.

PUBLICATIONS

Arndt, J.W., Schwarzenbacher, R., Page, R., et al. Crystal structure of an / serine

hydrolase (YDR428C) from Saccharomyces cerevisiae at 1.85 Å resolution. Proteins

58:755, 2005.

Bakolitsa, C., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of an

orphan protein (TM0875) from Thermotoga maritima at 2.00-Å resolution reveals

a new fold. Proteins 56:607, 2004.

Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M.E., Alvarez, R., Bryan,

M.C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D.J., Skehel, J.J.,

van Die, I., Burton, D.R., Wilson, I.A., Cummings, R., Bovin, N., Wong, C.H.,

Paulson, J.C. Printed covalent glycan array for ligand profiling of diverse glycan

binding proteins. Proc. Natl. Acad. Sci. U. S. A. 101:17033, 2004.

Bryan, M.C., Fazio, F., Lee, H.K., Huang, C.Y., Chang, A., Best, M.D., Calarese,

D.A., Blixt, O., Paulson, J.C., Burton, D., Wilson, I.A., Wong, C.-H. Covalent display

of oligosaccharide arrays in microtiter plates. J. Am. Chem. Soc. 126:8640, 2004.

Canaves, J.M., Page, R., Wilson, I.A., Stevens, R.C. Protein biophysical properties

that correlate with crystallization success in Thermotoga maritima: maximum clustering

strategy for structural genomics. J. Mol. Biol. 344:977, 2004.

Cardoso, R.M., Zwick, M.B., Stanfield, R.L., Kunert, R., Binley, J.M., Katinger, H.,

Burton, D.R., Wilson, I.A. Broadly neutralizing anti-HIV antibody 4E10 recognizes

a helical conformation of a highly conserved fusion-associated motif in gp41.

Immunity 22:163, 2005.

Crispin, M.D., Ritchie, G.E., Critchley, A.J., Morgan, B.P., Wilson, I.A., Dwek, R.A., Sim,

R.B., Rudd, P.M. Monoglucosylated glycans in the secreted human complement component

C3: implications for protein biosynthesis and structure. FEBS Lett. 566:270, 2004.

Debler, E.W., Ito, S., Seebeck, F.P., Heine, A., Hilvert, D., Wilson, I.A. Structural

origins of efficient proton abstraction from carbon by a catalytic antibody. Proc.

Natl. Acad. Sci. U. S. A. 102:4984, 2005.

Foss, T.R., Kelker, M.S., Wiseman, R.L., Wilson, I.A., Kelly, J.W. Kinetic stabilization

of the native state by protein engineering: implications for inhibition of transthyretin

amyloidogenesis. J. Mol. Biol. 347:841, 2005.

Han, G.W., Schwarzenbacher, R., Page, R., et al. Crystal structure of an alanineglyoxylate

aminotransferase from Anabaena sp at 1.70 Å resolution reveals a noncovalently

linked PLP cofactor. Proteins 58:971, 2005.

Hava, D.L., Brigl, M., van den Elzen, P., Zajonc, D.M., Wilson, I.A., Brenner,

M.B. CD1 assembly and the formation of CD1-antigen complexes. Curr. Opin.

Immunol. 17:88, 2005.

Heine, A., Canaves, J.M., von Delft, F., et al. Crystal structure of O-acetylserine

sulfhydrylase (TM0665) from Thermotoga maritima at 1.8 Å resolution. Proteins

56:387, 2004.

Heine, A., Luz, J.G., Wong, C.H., Wilson, I.A. Analysis of the class I aldolase

binding site architecture based on the crystal structure of 2-deoxyribose-5-phosphate

aldolase at 0.99 Å resolution. J. Mol. Biol. 343:1019, 2004.

Jaroszewski, L., Schwarzenbacher, R., von Delft, F., et al. Crystal structure of a

novel manganese-containing cupin (TM1459) from Thermotoga maritima at 1.65 Å

resolution. Proteins 56:611, 2004.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Kelker, M.S., Debler, E.W., Wilson, I.A. Crystal structure of mouse triggering

receptor expressed on myeloid cells 1 (TREM-1) at 1.76 Å. J. Mol. Biol.

344:1175, 2004.

Kelker, M.S., Foss, T.R., Peti, W., Teyton, L., Kelly, J.W., Wüthrich, K., Wilson,

I.A. Crystal structure of human triggering receptor expressed on myeloid cells 1

(TREM-1) at 1.47 Å. J. Mol. Biol. 342:1237, 2004.

Larsen, N.A., de Prada, P., Deng, S.X., Mittal, A., Braskett, M., Zhu, X., Wilson,

I.A., Landry, D.W. Crystallographic and biochemical analysis of cocaine-degrading

antibody 15A10. Biochemistry 43:8067, 2004.

Levin, I., Miller, M.D., Schwarzenbacher, R., et al. Crystal structure of an indigoidine

synthase A (IndA)-like protein (TM1464) from Thermotoga maritima at 1.90 Å

resolution reveals a new fold. Proteins 59:864, 2005.

Levin, I., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a putative

NADPH-dependent oxidoreductase (GI: 18204011) from mouse at 2.10 Å resolution.

Proteins 56:629, 2004.

Levin, I., Schwarzenbacher, R., Page, R., et al. Crystal structure of a PIN (PilT

N-terminus) domain (AF0591) from Archaeoglobus fulgidus at 1.90 Å resolution.

Proteins 56:404, 2004.

Li, C., Xu, L., Wolan, D.W., Wilson, I.A., Olson, A.J. Virtual screening of human

5-aminoimidazole-4-carboxamide ribonucleotide transformylase against the NCI

diversity set by use of AutoDock to identify novel nonfolate inhibitors. J. Med.

Chem. 47:6681, 2004.

Mathews, I., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of

S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) from Thermotoga

maritima at 2.0 Å resolution reveals a new fold. Proteins 59:869, 2005.

McMullan, D., Schwarzenbacher, R., Hodgson, K.O., et al. Crystal structure of a

novel Thermotoga maritima enzyme (TM1112) from the cupin family at 1.83 Å

resolution. Proteins 56:615, 2004.

Miller, M.D., Schwarzenbacher, R., von Delft, F., et al. Crystal structure of a tandem

cystathionine-β-synthase (CBS) domain protein (TM0935) from Thermotoga

maritima at 1.87 Å resolution. Proteins 57:213, 2004.

Page, R., Peti, W., Wilson, I.A., Stevens, R.C., Wüthrich, K. NMR screening and

crystal quality of bacterially expressed prokaryotic and eukaryotic proteins in a

structural genomics pipeline. Proc. Natl. Acad. Sci. U. S. A. 102:1901, 2005.

Pantophlet, R., Wilson, I.A., Burton, D.R. Improved design of an antigen with

enhanced specificity for the broadly HIV-neutralizing antibody b12. Protein Eng.

Des. Sel. 17:749, 2004.

Reiser, J.B., Teyton, L., Wilson, I.A. Crystal structure of the Drosophila peptidoglycan

recognition protein (PGRP)-SA at 1.56 Å resolution. J. Mol. Biol. 340:909, 2004.

Santelli, E., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a glycerophosphodiester

phosphodiesterase (GDPD) from Thermotoga maritima (TM1621)

at 1.60 Å resolution. Proteins 56:167, 2004.

Schwarzenbacher, R., Jaroszewski, L., von Delft, F., et al. Crystal structure of an

aspartate aminotransferase (TM1255) from Thermotoga maritima at 1.90 Å resolution.

Proteins 55:759, 2004.

Schwarzenbacher, R., Jaroszewski, L., von Delft, F., et al. Crystal structure of a

type II quinolic acid phosphoribosyltransferase (TM1645) from Thermotoga maritima

at 2.50 Å resolution. Proteins 55:768, 2004.

Schwarzenbacher, R., von Delft, F., Jaroszewski, L., et al. Crystal structure of a

putative oxalate decarboxylase (TM1287) from Thermotoga maritima at 1.95 Å

resolution. Proteins 56:392, 2004.

Spraggon, G., Pantazatos, D., Klock, H.E., Wilson, I.A., Woods, V.L., Jr., Lesley,

S.A. On the use of DXMS to produce more crystallizable proteins: structures of the

T maritima proteins TM0160 and TM1171 [published correction appears in Protein

Sci. 14:1688, 2005]. Protein Sci. 13:3187, 2004.

Spraggon, G., Schwarzenbacher, R., Kreusch, A., et al. Crystal structure of a methionine

aminopeptidase (TM1478) from Thermotoga maritima at 1.9 Å resolution.

Proteins 56:396, 2004.


Spraggon, G., Schwarzenbacher, R., Kreusch, A., et al. Crystal structure of a Udpn-acetylmuramate-alanine

ligase MurC (TM0231) from Thermotoga maritima at

2.3 Å resolution. Proteins 55:1078, 2004.

Stanfield, R.L., Dooley, H., Flajnik, M.F., Wilson, I.A. Crystal structure of a shark single-domain

antibody V region in complex with lysozyme. Science 305:1770, 2004.

Wang, X., Matteson, J., An, Y., Moyer, B., Yoo, J.S., Bannykh, S., Wilson, I.A., Riordan,

J.R., Balch, W.E. COPII-dependent export of cystic fibrosis transmembrane conductance

regulator from the ER uses a di-acidic exit code. J. Cell Biol. 167:65, 2004.

Xu, L., Li, C., Olson, A.J., Wilson, I.A. Crystal structure of avian aminoimidazole-

4-carboxamide ribonucleotide transformylase in complex with a novel non-folate

inhibitor identified by virtual ligand screening. J. Biol. Chem. 279:50555, 2004.

Xu, Q., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a formiminotetrahydrofolate

cyclodeaminase (TM1560) from Thermotoga maritima at 2.80 Å

resolution reveals a new fold. Proteins 58:976, 2005.

Xu, Q., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a ribose-5phosphate

isomerase RpiB (TM1080) from Thermotoga maritima at 1.90 Å resolution.

Proteins 56:171, 2004.

Xu, Q., Schwarzenbacher, R., Page, R., et al. Crystal structure of an allantoicase

(YIR029W) from Saccharomyces cerevisiae at 2.4 Å resolution. Proteins 56:619, 2004.

Zajonc, D.M., Crispin, M.D., Bowden, T.A., Young, D.C., Cheng, T.Y., Hu, J., Costello,

C.E., Rudd, P.M., Dwek, R.A., Miller, M.J., Brenner, M.B., Moody, D.B., Wilson, I.A.

Molecular mechanism of lipopeptide presentation by CD1a. Immunity 22:209, 2005.

Zhu, X., Tanaka, F., Hu, Y., Heine, A., Fuller, R., Zhong, G., Olson, A.J., Lerner,

R.A., Barbas, C.F. III, Wilson, I.A. The origin of enantioselectivity in aldolase antibodies:

crystal structure, site-directed mutagenesis, and computational analysis. J.

Mol. Biol. 343:1269, 2004.

Structure and Function of

Proteins as Molecular Machines

E.D. Getzoff, M. Aoyagi, A.S. Arvai, D.P. Barondeau,

R.M. Brudler, T. Cross, E.D. Garcin, C. Hitomi, K. Hitomi,

L. Holden, C.J. Kassmann, I. Li, M.E. Pique, M.E. Stroupe,

J.L. Tubbs, T.I. Wood

Our goals are to understand how proteins function

as molecular machines. We use structural,

molecular, and computational biology to study

proteins of biological and biomedical interest, especially

proteins that work synergistically with coupled

chromophores, metal ions, or other cofactors.

PHOTOACTIVE PROTEINS AND CIRCADIAN CLOCKS

To understand in atomic detail how proteins translate

sunlight into defined conformational changes for

biological functions, we are exploring the reaction mechanisms

of the blue-light receptors photoactive yellow

protein (PYP), photolyase, and cryptochrome. PYP is

the prototype for the Per-Arnt-Sim domain proteins of

circadian clocks, whereas proteins of the photolyase

and cryptochrome family catalyze DNA repair or act in

circadian clocks. To understand the protein photocycle

(Fig. 1), we combined our ultra-high-resolution and

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MOLECULAR BIOLOGY 2005 167

Fig. 1. Changes in the flexibility and mobility of PYP during its

light cycle revealed by mapping the results of hydrogen-deuterium

exchange mass spectrometry analyses (gray-scale shading) onto the

x-ray crystallographic structure (ribbon showing overall protein fold).

In the signaling state, regions of the protein including the N terminus

are released for protein-protein interactions.

time-resolved crystallographic structures of the dark

state and 2 photocycle intermediates of PYP with sitedirected

mutagenesis; ultraviolet-visible spectroscopy;

time-resolved Fourier transform infrared spectroscopy;

deuterium hydrogen exchange mass spectrometry, in

collaboration with V. Woods, University of California,

San Diego; and quantum mechanical and electrostatic

computational methods, in collaboration with L. Noodleman,

Department of Molecular Biology.

Cryptochrome flavoproteins are homologs of lightdependent

DNA repair photolyases that function as

blue-light receptors in plants and as components of

circadian clocks in animals. We determined the first

crystallographic structure of a cryptochrome, which

revealed commonalities with photolyases in DNA binding

and redox-dependent function but showed differences

in active-site and interaction surface features. New

structures of photolyases from 2 other branches of the

photolyase/cryptochrome family that repair cyclobutane

pyrimidine dimers and photoproducts helped us decipher

the cryptic structure, function, and evolutionary

relationships of these fascinating redox-active proteins.

A simple, but functional, circadian clock can be

reconstituted in vitro from the 3 cyanobacterial proteins

KaiA, KaiB, and KaiC alone. Yet, the structure

and dynamics of the functional assembly of these proteins

are not understood. Our crystallographic, dynamical

light scattering and small-angle x-ray scattering

studies revealed that KaiB self-assembles into a tetramer

(Fig. 2). We are also studying clock proteins with

PYP-like and Per-Arnt-Sim domains that bind to mammalian

cryptochromes. Our goal is to determine the

detailed chemistry and atomic structure of these pro-


168 MOLECULAR BIOLOGY 2005

Fig. 2. The tetrameric assembly of the cyanobacterial circadian

clock protein KaiB revealed by small-angle x-ray scattering (experimentally

determined shape) and x-ray crystallography (ribbon showing

protein fold).

teins, define their mechanisms of action and interaction,

and use our results to understand and regulate

biological function.

METALLOENZYME STRUCTURE AND FUNCTION

Superoxide dismutases (SODs) act as master regulators

of intracellular free radicals and reactive oxygen

species by transforming superoxide to oxygen and

hydrogen peroxide. Novel nickel SODs assemble into

hollow spheres composed of six 4-helix bundle subunits.

The 9 N-terminal residues fold into a unique

nickel hook motif that shows promise as a detectable

metal ion–binding tag in protein purification and structure

determination.

Our crystallographic structures of classic copper-zinc

SODs from mammals, bacterial symbionts, and pathogens

revealed striking differences in the enzyme assembly

and in the loops flanking the active-site channel,

despite the shared β-barrel subunit fold, catalytic

metal center, and electrostatic enhancement of activity.

With J. Tainer, Department of Molecular Biology,

we determined structures of mutant human SODs

found in patients with the disease amyotrophic lateral

sclerosis (Lou Gehrig disease), and proposed a hypothesis

for how single-site mutations cause this fatal neurodegenerative

disease.

To synthesize nitric oxide, a cellular signal and defensive

cytotoxin, nitric oxide synthases (NOSs) require calmodulin-orchestrated

interactions between their catalytic,

heme-containing oxygenase module and their electronsupplying

reductase module. Crystallographic structures

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

of wild-type and mutant NOS oxygenase dimers with

substrate, intermediate, inhibitors, cofactors, and cofactor

analogs, determined in collaboration with D. Stuehr,

the Cleveland Clinic, Cleveland, Ohio, and J. Tainer,

provided insights into the catalytic mechanism and

dimer stability.

Our structure-based drug design projects are aimed

at selectively inhibiting inducible NOS, to prevent inflammatory

disorders, or neuronal NOS, to prevent migraines,

while maintaining blood pressure regulation by endothelial

NOS. We integrated biochemical data with our

structures of NOS oxygenase, NOS reductase, and calmodulin

in complex with peptides derived from NOS

to propose a model for the assembled holoenzyme that

provides a moving-domain mechanism for electron flow

from NADPH through 2 flavin cofactors to the heme.

Our structure of the NOS reductase provides new

insights into the complex regulatory mechanisms of

this enzyme family.

METALLOPROTEIN DESIGN

An ultimate goal for protein engineers is to design

and construct new protein variants with desirable catalytic

or physical properties. As members of the Scripps

Research Metalloprotein Structure and Design Group,

we are testing our understanding of the affinity, selectivity,

and activity of metal ions by transplanting metal

sites from structurally characterized metalloproteins into

new protein scaffolds. To aid our design efforts, we have

organized quantitative information and interactive viewing

of protein metal sites at the Metalloprotein Database

and Browser (available at http://metallo.scripps.edu).

For green fluorescent protein and the homologous

red fluorescent protein, we designed, constructed, and

characterized metal-ion biosensors in which binding of

metal ions is signaled by changes in the spectroscopic

properties of the naturally occurring fluorophores. The

green fluorescent protein scaffold provides advantages

over existing probes by allowing optimization with random

mutagenesis, noninvasive expression in living cells,

and targeting to specific cellular locations. By completing

the metalloprotein design cycle from prediction to

highly accurate structures, we can rigorously evaluate

and improve our algorithms for the design of metal sites.

Our related structural studies of green and red fluorescent

protein intermediates in chromophore cyclization

and oxidation provide a novel mechanism for the spontaneous

synthesis of these tripeptide fluorophores within

the protein scaffold.


PUBLICATIONS

Barondeau, D.P., Getzoff, E.D. Structural insights into protein-metal ion partnerships.

Curr. Opin. Struct. Biol. 14:765, 2004.

Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Understanding GFP

chromophore biosynthesis: controlling backbone cyclization and modifying posttranslational

chemistry. Biochemistry 44:1960, 2005.

Dunn, A.R., Belliston-Bittner, W., Winkler, J.R., Getzoff, E.D., Stuehr, D.J., Gray,

H.B. Luminescent ruthenium(II)- and rhenium(I)-diimine wires bind nitric oxide

synthase. J. Am. Chem. Soc. 127:5169, 2005.

Hitomi, K., Oyama, T., Han, S., Arvai, A.S., Getzoff, E.D. Tetrameric architecture

of the circadian clock protein KaiB: a novel interface for intermolecular interactions

and its impact on the circadian rhythm. J. Biol. Chem. 280:19127, 2005.

Stroupe, M.E., Getzoff, E.D. The role of siroheme in sulfite and nitrite reductases.

In: Tetrapyrroles: Their Birth, Life and Death. Warren, M.J., Smith, A. (Eds.). Landes

Bioscience, Georgetown, Tex, in press.

Stuehr, D.J., Wei, C.C., Santolini, J., Wang, Z., Aoyagi, M., Getzoff, E.D. Radical

reactions of nitric oxide synthases. In: Free Radicals: Enzymology, Signaling, and

Disease. Cooper, C.E., Wilson, M.T., Darley-Usmar, V.H. (Eds.). Portland Press,

London, 2004, p. 39. Biochemical Society Symposia, Vol. 71.

Tiso, M., Konas, D.W., Panda, K., Garcin, E.D., Sharma, M., Getzoff, E.D., Stuehr, D.J.

C-terminal tail residue ARG1400 enables NADPH to regulate electron transfer in

neuronal nitric oxide synthase. J. Biol. Chem., in press.

Tubbs, J.L., Tainer, J.A., Getzoff, E.D. Crystallographic structures of Discosoma

red fluorescent protein with immature and mature chromophores: linking peptide

bond trans-cis isomerization and acylimine formation in chromophore maturation.

Biochemistry 44:9833, 2005.

Vevodova, J., Graham, R.M., Raux, E., Schubert, H.L., Roper, D.I., Brindley,

A.A., Scott, A.I., Roessner, C.A., Stamford, N.P., Stroupe, M.E., Getzoff, E.D.,

Warren, M.J., Wilson, K.S. Structure/function studies on an S-adenosyl-L-methionine-dependent

uroporphyrinogen III C methyltransferase (SUMT), a key regulatory

enzyme of tetrapyrrole biosynthesis. J. Mol. Biol. 344:419, 2004.

Wei, C.C., Wang, Z.Q., Durra, D., Hemann, C., Hille, R., Garcin, E.D., Getzoff,

E.D., Stuehr, D.J. The three nitric-oxide synthases differ in their kinetics of tetrahydrobiopterin

radical formation, heme-dioxy reduction, and arginine hydroxylation. J.

Biol. Chem. 280:8929, 2005.

Structural Molecular Biology of

Interactions and Protein Design

J.A. Tainer, A.S. Arvai, D.P. Barondeau, M. Bjoras,

B.R. Chapados, L. Craig, T.H. Cross, D.S. Daniels, G. DiVita,

L. Fan, C. Hitomi, K. Hitomi, J.L. Huffman, C.J. Kassmann,

I. Li, G. Moncalian, M.E. Pique, D.S. Shin, O. Sundheim,

R.S. Williams, T.I. Wood, A. Yamagata

Our goals are to bridge the gap between the vastly

improved tools and insights for structural cell

biology at the molecular level and the applications

of these advances for the molecular-based understanding

of and eventual intervention in human diseases.

Thus, our primary concern is the application of structural

biology to fundamental questions of molecular and

cellular biology relevant to human disease. Currently,

we are investigating fundamental processes and principles

of DNA repair, control of reactive oxygen species,

control of the cell cycle, and pathogenesis. We think

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The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 169

these processes have networked connections and common

themes in terms of structural mechanisms and

controls and medical implications. In general, our

structural determination and design work involves

hypothesis-driven studies; we focus on high-resolution

structural analyses, functionally important conformational

changes, and macromolecular interactions,

including design of inhibitors and dynamic assemblies

that act as macromolecular machines to control the

fundamental processes of cell biology.

To accomplish our basic research, we use protein

crystallography, solution x-ray scattering, fluorescence,

biochemistry, mutagenesis, and protein expression. Our

experimental work is complemented by efforts to develop

new methods, particularly in structural analysis, protein

and drug design, and the merging of crystal structures

with x-ray solution structures and electron microscopy.

These new experimental integrations involve the use of

synchrotron radiation to bridge the size and resolution

gap between high-resolution macromolecular structures

and the multiprotein macromolecular machines and

reversible interactions in the cell. For protein design,

we have an active collaboration with E. Getzoff, Department

of Molecular Biology, to understand and control

the formation of self-synthesizing chromophores in green

fluorescent protein and its homologs. We are increasingly

interested in structure-based design of inhibitors

that are relevant to the development of novel therapeutic

agents and inhibitors that chemically knock out or block

gene function to complement genes that are knocked

out by removing the DNA. The synergy between basic

research and advances in techniques is allowing us to

contribute to the basic understanding and treatment of

degenerative and infectious diseases and cancer.

SUPEROXIDE DISMUTASES

Superoxide dismutases (SODs) are master regulators

for reactive oxygen species involved in injury, pathogenesis,

aging, and degenerative diseases. In basic

research on these enzymes, we are characterizing the

activity of the mitochondrial SODs. We discovered a

novel nickel ion SOD and characterized its hexameric

assembly. For the human cytoplasmic copper, zinc SOD,

we examined how single-site mutations cause the neurodegenerative

Lou Gehrig disease or familial amyotrophic

lateral sclerosis (FALS). We found that point mutations

destabilize the copper, zinc SOD dimer and dramatically

increase its propensity to aggregate and form filaments

that resemble those seen in motor neurons of

patients with FALS. These findings provide a molecu-


170 MOLECULAR BIOLOGY 2005

lar basis for the notion that a single FALS disease phenotype

arises from diverse point mutations throughout

the protein that reduce the structural integrity of

copper, zinc SOD and lower the energy barrier for fibrous

aggregation. Additionally, our new high-resolution structures

of a related thermophilic copper, zinc SOD showed

a trapped product complex. This novel finding helps

define the enzyme’s mechanism of action and its susceptibility

to inactivation by hydrogen peroxide.

DNA REPAIR

All life requires constant repair of DNA. Structural

and mutational analyses of DNA repair enzymes provide

a framework for understanding the molecular basis

of genetic integrity and the loss of this integrity in cancer

and degenerative diseases. We are interested in how specific

types of damage are detected, how repair enzymes

are coordinated within different pathways, and the

nature and role of conformational change in proteins

and DNA in repair pathways. We use electron microscopy,

x-ray crystallography, small-angle x-ray scattering,

and complementary in vitro and in vivo mutational

analysis to go from enzyme structures to repair pathways

and the coordination of repair with replication

and transcription.

We focus on pathways for DNA base repair, DNA

nick translation in repair and replication (Fig. 1), and

repair of double-stranded breaks. Understanding the

structural chemistry and cell biology of DNA repair is

critical for designing specific inhibitors to increase the

effectiveness of chemotherapy and also for assessing

how DNA repair enzyme polymorphisms may affect

diseases in humans. Currently, we are designing inhibitors

of enzymes that repair alkylated and oxidized guanines.

These enzymes are one of the body’s natural

defenses against DNA damage, but they can also inadvertently

protect cancer cells from chemotherapeutic

agents. For example, the human repair protein O 6 -alkylguanine-DNA

alkyltransferase, which acts in the repair

of alkylated guanines, repairs damaged DNA inside

human cells, and cancer cells can use it to repair DNA

that has been damaged in the course of chemotherapy,

thus making the chemotherapy ineffective.

BACTERIAL PILI

Type IV pili are essential virulence factors for many

gram-negative bacteria, playing key roles in surface

motility, adhesion, formation of microcolonies and biofilms,

natural transformation, and signaling. We have

determined structures for the type IV pilin subunits and

for the assembled pilus fiber. Currently, we are investigating

the type IV pilus assembly system, including the

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Fig. 1. Interactions between the complex consisting of flap endonuclease

1 (FEN-1), DNA, and proliferating cell nuclear antigen

(PCNA) and the interface of DNA repair and replication. A, Nicked

DNA is protected and repaired by the sequential activities of DNA

polymerase δ (pol δ) and FEN-1 held to DNA by the "sliding clamp"

PCNA. In the absence of FEN-1, a complex of pol δ and PCNA binds

to and protects the nick (top). FEN-1 initiates nick translation by

binding to PCNA (bottom), recognizing the 3′ DNA flap and cleaving

the 5′ flap, generating a nick translated by 1 nucleotide. B. Structures

of FEN-1 bound to DNA show that FEN-1 recognizes the 3′ flap

in a sequence-independent manner. C, A composite model of the

FEN-1–DNA–PCNA complex suggests how a kinked DNA intermediate

might facilitate sequential activities of FEN-1 and pol δ.

assembly ATPase, the membrane anchor protein interactions,

and the assembled pilus fiber (Fig. 2). Our electron

Fig. 2. A schematic view of the assembly machinery of type IV

pili: the electron cryomicroscopy structure of the pilus of Neisseria

gonorrhoeae (GC); crystal structures of full-length Pseudomonas

aeruginosa (P.a) pilin; BfpC, the binding partner protein to ATPase

from enteropathogenic Escherichia coli; and GspE2, the hexameric

assembly ATPase from Archaeoglobus fulgidus.


microscopy and x-ray structures of protein components

and complexes are helping us understand the architecture

and assembly mechanism as a basis for the design

of antibacterial vaccines and therapeutic agents.

PUBLICATIONS

Ayala, I., Perry, J.P., Szczepanski, J., Tainer, J.A., Vala, M.T., Nick, H.S., Silverman,

D.N. Hydrogen bonding in human manganese superoxide dismutase containing

3-fluorotyrosine. Biophys. J., in press.

Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Understanding GFP

chromophore biosynthesis: controlling backbone cyclization and modifying posttranslational

chemistry. Biochemistry 44:1960, 2005.

Crowther, L.J., Yamagata, A., Craig, L., Tainer, J.A., Donnenberg, M.S. The ATPase

activity of BfpD is greatly enhanced by zinc and allosteric interactions with other

Bfp proteins. J. Biol. Chem. 280:24839, 2005.

de Jager, M., Trujillo, K.M., Sung, P., Hopfner, K.P., Carney, J.P., Tainer, J.A.,

Connelly, J.C., Leach, D.R., Kanaar, R., Wyman, C. Differential arrangements of

conserved building blocks among homologs of the Rad50/Mre11 DNA repair protein

complex. J. Mol. Biol. 339:937, 2004.

Garcin, E.D., Bruns, C.M., Lloyd, S.J., Hosfield, D.J., Tiso, M., Gachhui, R.,

Stuehr, D.J., Tainer, J.A., Getzoff, E.D. Structural basis for isozyme-specific regulation

of electron transfer in nitric-oxide synthase. J. Biol. Chem. 279:37918, 2004.

Hendrickson, E.A., Huffman, J.L., Tainer, J.A. Structural aspects of Ku and the

DNA-dependent protein kinase complex. In: DNA Damage Recognition. Seide, W.,

Kow, Y.W., Doetsch, P.W. (Eds.). Taylor & Francis, New York, 2005, p. 629.

Huffman, J.L., Sundheim, O., Tainer, J.A. DNA base damage recognition and

removal: new twists and grooves. Mutat. Res. 577:55, 2005.

Huffman, J.L., Sundheim, O., Tainer, J.A. Structural features of DNA glycosylases

and AP endonucleases. In: DNA Damage Recognition. Seide, W., Kow, Y.W.,

Doetsch, P.W. (Eds.). Taylor & Francis, New York, 2005, p. 299.

Manuel, R.C., Hitomi, K., Arvai, A.S., House, P.G., Kurtz, A.J., Dodson, M.L., McCullough,

A.K., Tainer, J.A., Lloyd, R.S. Reaction intermediates in the catalytic mechanism

of Escherichia coli MutY DNA glycosylase. J. Biol. Chem. 279:46930, 2004.

Putnam, C.D.. Tainer, J.A. Protein mimicry of DNA and pathway regulation. DNA

Repair (Amst.), in press.

Sarker, A.H., Tsutakawa, S.E., Kostek, S., Ng, C., Shin, D.S., Peris, M., Campeau, E.,

Tainer, J.A., Nogales, E., Cooper, P.K. Recognition of RNA polymerase II and transcription

bubbles by XPG, CSB, and TFIIH: insights for transcription-coupled repair

and Cockayne syndrome. Mol. Cell 20:187, 2005.

Simeoni, F., Arvai, A., Bello, P., Gondeau, C., Hopfner, K.P., Neyroz, P., Heitz, F.,

Tainer, J., Divita, G. Biochemical characterization and crystal structure of a Dim1

family associated protein: Dim2. Biochemistry 44:11997, 2005.

Tubbs, J.L., Tainer, J.A., Getzoff, E.D. Crystallographic structures of Discosoma

red fluorescent protein with immature and mature chromophores: linking peptide

bond trans-cis isomerization and acylimine formation in chromophore maturation.

Biochemistry 44:9833, 2005.

Williams, R.S., Tainer, J.A. A nanomachine for making ends meet: MRN is a flexing

scaffold for the repair of DNA double-strand breaks. Mol. Cell 19:724, 2005.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 171

Structural Biology of Integral

Membrane Proteins

G. Chang, A. Chen, Y. Chen, X. He, O. Pornillos, C.R. Reyes,

P. Szewczk, A. Ward, S. Wada, Y. Yin

X-ray crystallography of integral membrane proteins

is an exciting and rapidly growing frontier in

molecular structural biology. We are interested in

5 areas: (1) the molecular structural basis for lipid and

drug transport across the cell membrane by multidrugresistance

(MDR) transporters, (2) the high-resolution

structure of yeast and mammalian MDR transporters,

(3) signal transduction by receptors, (4) discovery and

the structurally based design of potent MDR reversal

agents, and (5) the development of an in vitro cell-free

system capable of overproducing integral membrane proteins

suitable for biophysical study. We use several experimental

methods, including detergent/lipid protein

biochemistry, 3-dimensional crystallization of integral

membrane proteins, and x-ray crystallography. We are

developing and using an efficient cell-free membrane protein

expression system in collaboration with T. Kudlicki,

Invitrogen Corporation, Carlsbad, California, for the overexpression

integral membrane proteins for both x-ray

crystallography and nuclear magnetic resonance studies.

We are addressing the molecular basis of MDR, a

significant challenge in the treatment of infectious disease

and cancer. A major cause of MDR in both of these

situations is a battery of drug efflux pumps imbedded

in the cell membrane. Through our structural studies

on MDR transporters, we hope to gain insights into

the mechanics of translocating amphipathic substrates

across the cell membrane and also the rational design

of potent MDR reversal agents.

We are combining chemistry and biology with structure

for the discovery and design of potent MDR reversal

agents for cancer chemotherapy in collaboration with

M.G. Finn, Department of Chemistry; I. Urbatsch, Texas

Tech University Health Sciences Center, Lubbock Texas;

and S. Reutz, Novartis International AG, Basel, Switzerland.

In collaboration with M. Saier, University of California,

San Diego, and Q. Zhang, Department of Molecular

Biology, we are determining the x-ray structures and

mapping the detailed functional components of 3 families

of bacterial MDR transporters that are dominant

in gram-positive pathogens. In another collaboration,

with R.A. Milligan, Department of Cell Biology, we are

using electron cryomicroscopy to visualize the low-res-


172 MOLECULAR BIOLOGY 2005

olution structures of our transporters. Through these

united efforts, we will gain a broader understanding of

the structure and function of drug transporters that

cause MDR in cancer and bacterial infection.

Recently, we determined a new structure of the MDR

ATP-binding cassette transporter homolog MsbA in complex

with magnesium, adenosine diphosphate, inorganic

vanadate, and rough-chemotype lipopolysaccharide.

This structure supports a model involving a rigid-body

torque of the 2 transmembrane domains during ATP

hydrolysis and suggests a mechanism by which the

nucleotide-binding domain communicates with the transmembrane

domain. We propose a lipid “flip-flop” mechanism

in which the sugar groups are sequestered in

the chamber while the hydrophobic tails are dragged

through the lipid bilayer (Fig. 1). This posthydrolysis

Fig. 1. Proposed model for sequestering the polar sugar headgroup

of lipopolysaccharide (LPS) in the internal chamber of MsbA (for

clarity, only 1 LPS is shown). A, LPS initially binds to the elbow

helix as modeled onto the closed apo structure. B, Lipid headgroups

modeled to insert into the chamber of the apo closed structure.

C, As the transporter undergoes conformational changes related

to binding and hydrolysis of ATP, the headgroup is “flipped” within

the polar chamber while the LPS hydrocarbon chains are freely exposed

and dragged through the lipid bilayer. Both LPS and MsbA conformations

are modeled. D, LPS is presented to the outer leaflet of the

membrane as observed in this structure. Reprinted with permission

from Reyes, C.L., Chang, G. Science 308:1028, 2005.

structure of MsbA also gives insight into the possible

drug-binding sites for a number of cancer compounds.

We are continuing our x-ray structural studies of the

small MDR transporter EmrE and of other families of

bacterial MDR transporters to better understand the

molecular basis of the drug-proton antiport. The x-ray

structures of MsbA and EmrE are excellent models for

drug efflux systems that confer MDR to cancer cells and

infectious microorganisms.

PUBLICATIONS

Ma, C., Chang, G. Crystallography of the integral membrane protein EmrE from

Escherichia coli. Acta Crystallogr. D Biol. Crystallogr. 60:2399, 2004.

Reyes, C.L., Chang, G. Structure of the ABC transporter MsbA in complex with

ADP•vanadate and lipopolysaccharide. Science 308:1028, 2005.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Structure and Function of

Membrane-Bound Enzymes

C.D. Stout, H. Heaslet, M. Yamaguchi, V. Sundaresan,

L. Hunsicker-Wang, J. Chartron

One focus of our research is the structure and

function of transhydrogenase, an essential

enzyme of respiration in mitochondria and bacteria.

Transhydrogenase couples proton translocation

across the membrane with hydride transfer between

cofactors bound to soluble domains. We are determining

the structure of the enzyme in its membrane-bound

conformation and are studying the structures of the

soluble domains. For studies of enzyme function, we

are using biochemical methods and mutagenesis. Structural

studies entail x-ray crystallography, electron microscopy

studies done in collaboration with M. Yeager and

B. Carragher, Department of Cell Biology, and nuclear

magnetic resonance experiments done in collaboration

with J. Dyson, Department of Molecular Biology.

In collaboration with E.F. Johnson, Department of

Molecular Biology, and J.R. Halpert, University of Texas

Medical Branch, Galveston, Texas, we are studying highresolution

crystal structures of mammalian cytochrome

P450s. The P450s are monooxygenases involved in

the biosynthesis and oxidation of lipophilic molecules,

and they specifically metabolize a wide range of exogenous

compounds and drugs. More than 60 genes for

P450s occur in the human genome. We are studying

high-resolution structures and drug-bound complexes

of the human P450s 2C8, 2C9, 2A6, 3A4, and 1A2

and the rabbit P450s 2B4 and 2C5.

In collaboration with J.A. Fee, Department of Molecular

Biology, we are studying the structure and mechanism

of cytochrome ba 3 oxidase, the terminal enzyme

of respiration responsible for the reduction of molecular

oxygen to water. The high-resolution crystal structure

of the enzyme from a thermophilic bacterium has

been determined (Fig. 1). Crystallographic experiments,

in combination with mutagenesis and spectroscopy, are

being used to capture intermediates in the reaction

cycle and to define the pathways of proton translocation

to and from the active site within the membrane.

In parallel with these studies, we are developing

the application of nanodiscs for biophysical studies of

integral membrane proteins. These experiments are

being done in collaboration with S.G. Sligar, University

of Illinois, Urbana, Illinois, and M. Yeager, Department


Fig. 1. Crystal structure of the integral membrane protein cytochrome

ba3 oxidase from the thermophilic bacterium Thermus

thermophilus. Cytochrome oxidase is responsible for the reduction

of oxygen to water during respiration in all higher organisms.

of Cell Biology. Nanodiscs are water-soluble particles

that consist of 2 copies of an engineered construct of

human apolipoprotein A-I (~200 amino acids) encircling

a patch of bilayer containing the approximately 160

molecules of dimyristoyl-sn-glycero-3-phosphocholine

or other phospholipids. Integral membrane proteins

can be inserted into these particles by spontaneous

self-assembly, and to date we have incorporated both

cytochrome ba 3 oxidase and transhydrogenase.

Additional research projects involve collaboration

with other faculty members at Scripps Research. These

projects include studies of iron-sulfur and electron transfer

proteins, in collaboration with J.A. Fee and L. Noodleman,

Department of Molecular Biology; RNA-protein

complexes, with J.R. Williamson, Department of Molecular

Biology; synthetic, self-assembling peptides, with

M.R. Ghadiri, Department of Chemistry; and HIV protease

inhibitor complexes, with A. Olson, Department

of Molecular Biology, and B.E. Torbett, Department of

Molecular and Experimental Medicine.

PUBLICATIONS

Carroll, K.S., Gao, H., Chen, H., Stout, C.D., Leary, J.A., Bertozzi, C.R. A conserved

mechanism for sulfonucleotide reduction. PloS Biol. 3:e250, 2005.

Fee, J.A., Todaro, T.R., Luna, E., Sanders, D., Hunsicker-Wang, L.M., Patel, K.M.,

Bren, K.L., Gomez-Moran, E., Hill, M.G., Ai, J., Loehr, T.M., Oertling, W.A.,

Williams, P.A., Stout, C.D., McRee, D., Pastuszyn, A. Cytochrome rC 552 , formed

during expression of the truncated, Thermus thermophilus cytochrome c 552 gene

in the cytoplasm of Escherichia coli, reacts spontaneously to form protein-bound,

2-formyl-4-vinyl (Spirographis) heme. Biochemistry 43:12162, 2004.

Hays, A.-M., Dunn, A.R., Chiu, R., Gray, H.B., Stout, C.D., Goodin, D.B. Conformational

states of cytochrome P450cam revealed by trapping of synthetic wires. J.

Mol. Biol. 344:455, 2004.

Horne, W.S., Yadav, M.K., Stout, C.D., Ghadiri, M.R. Heterocyclic peptide backbone

modifications in an α-helical coiled coil. J. Am. Chem. Soc. 126:15366, 2004.

Hunsicker-Wang, L.M., Pacoma, R.L., Chen, Y., Fee, J.A., Stout, C.D. A novel

cryoprotection scheme for enhancing diffraction of crystals of recombinant cytochrome

ba 3 oxidase from Thermus thermophilus. Acta Crystallogr. D Biol. Crystallogr.

61:340, 2005.

Stout, C.D. Cytochrome P450 conformational diversity. Structure (Camb.)

12:1921, 2004.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Sundaresan, V., Chartron, J., Yamaguchi, M., Stout, C.D. Conformational diversity

in NAD(H) and interacting transhydrogenase nicotinamide nucleotide binding

domains. J. Mol. Biol. 346:617, 2005.

Wester, M.R., Yano, J.K., Schoch, G.A., Yang, C., Griffin, K.J., Stout, C.D., Johnson,

E.F. The structure of human cytochrome P450 2C9 complexed with flurbiprofen

at 2.0-Å resolution. J. Biol. Chem. 279:35630, 2004.

Yadav, M.K., Redman, J.E., Leman, L.J., Alvarez-Gutierrez, J.M., Zhang, Y.,

Stout, C.D., Ghadiri, M.R. Structure-based engineering of internal cavities in

coiled-coil peptides. Biochemistry 44:9723, 2005.

Yano, J.K., Hsu, M.H., Griffin, K.J., Stout, C.D., Johnson, E.F. Structures of

human microsomal cytochrome P450 2A6 complexed with coumarin and

methoxsalen. Nat. Struct. Mol. Biol. 12:822, 2005.

Yano, J.K., Wester, M.R., Schoch, G.A., Griffin, K.J., Stout, C.D., Johnson, E.F.

The structure of human microsomal cytochrome P450 3A4 determined by x-ray

crystallography to 2.05-Å resolution. J. Biol. Chem. 279:38091, 2004.

Lipid Chemistry for Studies of

Integral Membrane Proteins

Q. Zhang, M.G. Finn,* X. Ma

* Department of Chemistry, Scripps Research

MOLECULAR BIOLOGY 2005 173

Integral membrane proteins float in the lipid bilayer

with their hydrophobic domains threaded through the

membrane and their hydrophilic domains extended

into the aqueous solution. These proteins are extremely

unstable outside the hydrophobic membrane bilayer, a

situation that makes their in vitro biophysical and structural

characterization difficult. An artificial environment

is therefore needed to stabilize the proteins in their

native state. We are attempting to synthesize new

amphiphilic molecules that can extract integral membrane

proteins from membranes and stabilize the proteins

for structural characterization.

Relatively few investigators have actually addressed

questions about the design of appropriate amphiphilic

molecules despite the extensive use of such molecules

in studies of membrane proteins. The criteria that we

apply to generate such amphiphilic molecules are based

on the physical properties of the molecules and on their

interactions with membrane proteins. Detergents that

self-assemble into micellar structures are universally

used to dissolve integral membrane proteins as single

particles to facilitate protein crystallization. We intend

to incorporate more hydrophobicity in the interior of

detergent micelles to improve the stability of the micelles

and consequently their ability to stabilize integral membrane

proteins. We accomplish this incorporation by

appending branches along the alkyl chains of detergents

and, most interestingly, by adding a short branch at the

interface between the hydrophobic tail and the hydro-


174 MOLECULAR BIOLOGY 2005

philic head. These branches may behave in 2 distinct

ways like small amphiphile additives successfully used

in crystallization of integral membrane proteins, thereby

decreasing the micellar radius and extruding water

from the hydrophobic core of the micelles.

The effect of these modifications on detergent micelle

properties and on the stabilization and crystallization of

integral membrane proteins is being investigated in collaboration

with members of the Center for Innovative

Membrane Protein Technologies of the Joint Center for

Structural Genomics at Scripps Research. We are also

interested in synthesizing additional novel amphiphilic

molecules, including peptides, fluorinated lipids, and polymers

that have special properties to facilitate the structural

and functional study of integral membrane proteins.

High-Throughput Structure-

Based Drug Discovery and

Structural Neurobiology

R.C. Stevens, E.E. Abola, A. Alexandrov, J.W. Arndt,

G. Asmar-Rovira, R. Benoit, F. Bi, M.H. Bracey, D. Carlton,

Q. Chai, J.C. Chappie, E. Chien, T. Clayton, B. Collins,

A. Gámez, M. Griffith, C. Grittini, M.A. Hanson, A. Houle,

J. Joseph, K. Masuda, B. McManus, K. Moy, M. Nelson,

R. Page, M.G. Patch, C. Roth, K. Saikatendu, V. Sridhar,

M. Straub, V. Subramanian, J. Velasquez, L. Wang, M. Yadav

HIGH-THROUGHPUT STRUCTURAL BIOLOGY

For the past several years, we have focused on

developing tools to change the field of structural

biology by accelerating the rate of determination

of protein structures, an endeavor that includes pioneering

microliter expression/purification for structural studies,

nanovolume crystallization, and automated image

collection. Applications of these technologies were initially

tested at the Joint Center for Structural Genomics

(http://www.jcsg.org), where we showed the power of the

new tools. In addition to the recent funding of the JCSG-2

as a second-phase production center of the National Institute

of General Medical Sciences, 2 new centers funded

by the National Institutes of Health have been spun off for

technologic innovations in structural biology. The first center

is called the Joint Center for Innovative Membrane

Protein Technologies (http://jcimpt.scripps.edu). Here, in

collaboration with G. Chang, S. Lesley, K. Wüthrich,

and Q. Zhang, Department of Molecular Biology; P. Kuhn

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

and M. Yeager, Department of Cell Biology; and M.G.

Finn, Department of Chemistry, we do research exclusively

on membrane proteins, including G protein–coupled

receptors. The second center is the Accelerated Technologies

Center for Gene to 3D Structure (http://www

.atcg3d.org). Here we are doing collaborative studies with

P. Kuhn, Department of Cell Biology, and researchers

from deCODE biostructures, Bainbridge Island, Washington;

Lyncean Technologies, Palo Alto, California; and

the University of Chicago, Chicago, Illinois. In the near

future, this center will build a synchrotron resource at

Scripps Research.

STRUCTURAL NEUROBIOLOGY

Although we have developed high-throughput methods

to accelerate the determination of protein structures,

our primary interest is using these tools to study the

chemistry and biology of neurotransmission and of diseases

that affect neurons. Our goals are to understand

how neuronal cells function on a molecular level and,

on the basis of that understanding, create new molecules

and materials that mimic neuronal signal transduction

and recognition. We use high-throughput protein crystallography

and biochemical methods to probe the structure

and function of molecules involved in neurotransmission

and neurochemistry.

F A TTY ACID AMIDE HYDROLASE

In collaboration with B.F. Cravatt, Department of

Cell Biology, we solved the structure of fatty acid amide

hydrolase (FAAH), a degradative integral membrane

enzyme responsible for setting intracellular levels of

endocannabinoids, to 2.8 Å. FAAH is intimately associated

with CNS signaling processes such as retrograde

synaptic transmission, a process that is also modulated

by the illicit substance δ 9 -tetrahydrocannabinol. FAAH is

a dimer capable of monotopic membrane insertion; it

has an active-site structure consistent with the capacity

for hydrolysis of hydrophobic fatty acid amides and

structural features amenable to structure-based drug

design. With our knowledge of the 3-dimensional structure,

we are trying to understand how the enzyme works

at a basic level and how it might be the basis for potential

drug discovery.

BIOSYNTHESIS OF NEUROTRANSMITTERS

For neuronal signal transduction, the presynaptic

cell synthesizes neurotransmitters that then traverse

the synaptic cleft. We are using the high-throughput

methods to determine the inclusive structures of complete

biochemical pathways. Specifically, we are interested

in determining the structures of all the enzymes


in the biosynthesis pathways of neurotransmitters in

order to understand the mechanistic details of each

individual enzymatic reaction at the atomic level. This

approach also allows us to determine the best path of

drug discovery in the areas of neurotransmitter biosynthesis

and catabolism.

Phenylalanine hydroxylase and tyrosine hydroxylase

initiate the first committed step in the biosynthesis of

the neurotransmitters dopamine, adrenaline, and noradrenaline,

and tryptophan hydroxylase catalyzes the

rate-determining step in the biosynthesis of serotonin.

Because of the importance of these neurotransmitters

in the proper functioning of the CNS, understanding

the molecular details involved in the catalysis and regulation

of these biosynthetic enzymes is crucial. We

determined the 3-dimensional structures for tyrosine

hydroxylase, tryptophan hydroxylase, and phenylalanine

hydroxylase, and we are uncovering specific mechanistic

details for these enzymes.

THERAPEUTIC AGENTS FOR TREATMENT OF

PHENYLKETONURIA

In addition to the basic hydroxylase enzymology

questions under investigation, recent clinical studies

suggest that some patients with the metabolic disease

phenylketonuria are responsive to (6R)-L-erythro-5,6,7,

8-tetrahydrobiopterin, the natural cofactor of phenylalanine

hydroxylase. We are doing studies to correlate

how structure can be used to predict which patients

with phenylketonuria most likely will respond to treatment

with this cofactor. Currently, the proprietary form

of the cofactor, Phenoptin, is entering phase 3 clinical

trials for the treatment of mild phenylketonuria. For

classical phenylketonuria, we are developing an enzyme

replacement therapeutic agent that is being tested in

animal models. The therapy is based on administration

of a modified form of phenylalanine ammonia lyase

discovered in our structural studies (Fig. 1). Last, we

are determining the structural basis of diseases caused

by several other enzymes involved in the biosynthesis of

neurotransmitters. Many of these disorders are rare or

occur during childhood.

NEUROTOXINS

The clostridial neurotoxins include tetanus toxin and

the 7 serotypes of botulinum toxin (Fig. 2). We are

determining the molecular events involved in the binding,

pore formation, translocation, and catalysis of botulinum

neurotoxin. Although botulinum toxin is most

known for its deadly effects, it is now being used

therapeutically to treat involuntary muscle disorders.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 175

Fig. 1. A, Crystal structure of phenylalanine ammonia lyase (PAL)

determined at 1.6-Å resolution. This protein structure was engineered

and chemically modified as a potential once-a-week injectable therapeutic

agent for treatment of phenylketonuria. B, ENU2 mice are

used as a model for phenylketonuria in preclinical studies. C and

D, Reduction in phenylalanine and immune response levels in ENU2

mice after the injection of PAL that has been chemically modified

(pegylated). These PEG-PAL formulations show promise as therapeutic

agents for treatment of phenylketonuria.

Fig. 2. Serotype structures of botulinum neurotoxin (BoNT), its

light chain (LC), and the closely related tetanus neurotoxin (TeNT).


176 MOLECULAR BIOLOGY 2005

Recently, we determined the structure of the 900-kD

complex form of the toxin, the 150-kD holotoxin form,

the catalytic domain, and the catalytic domain bound

to substrates and inhibitors. These structures are being

used to understand and redesign the toxin’s mechanism

of action and to determine additional therapeutic applications

of the toxin.

PUBLICATIONS

Arndt, J.W., Gu, J., Jaroszewski, L., Schwarzenbacher, R., Hanson, M.A.,

Lebeda, F.J., Stevens, R.C. The structure of the neurotoxin-associated protein

HA33/A from Clostridium botulinum suggests a reoccurring β-trefoil fold in the

progenitor toxin complex. J. Mol. Biol. 346:1083, 2005.

Arndt, J.W., Schwarzenbacher, R., Page, R., et al. Crystal structure of an α/β serine

hydrolase (YDR428C) from Saccharomyces cerevisiae at 1.85 Å resolution.

Proteins 58:755, 2005.

Arndt, J.W., Yu, W., Bi, F., Stevens, R.C. Crystal structure of botulinum neurotoxin

type G light chain: serotype divergence in substrate recognition. Biochemistry

44:9574, 2005.

Cànaves, J.M., Page, R., Stevens, R.C. Protein biophysical properties that correlate

with crystallization success in Thermotoga maritima: maximum clustering

strategy for structural genomics. J. Mol. Biol. 344:977, 2004.

Carter, D.C., Rhodes, P., McRee, D.E., Tari, L.W., Dougan, D.R., Snell, G., Abola, E.,

Stevens, R.C. Reduction in diffuso-convective disturbances in nanovolume protein

crystallization experiments. J. Appl. Crystrallogr. 38:87, 2005.

Chappie, J.S., Cànaves, J.M., Han, G.W., Rife, C.L., Xu, Q., Stevens, R.C. The

structure of a eukaryotic nicotinic acid phosphoribosyltransferase reveals structural

heterogeneity among type II PRTases. Structure (Camb.) 13:1385, 2005.

Erlandsen, H., Pey, A.L., Gámez, A., Pérez, B., Desviat, L.R., Aguado, C., Koch,

R., Surendran, S., Tyring, T., Matalon, R., Scriver, C.R., Ugarte, M., Martínez, A.,

Stevens, R.C. Correction of kinetic and stability defects by the cofactor tetrahydrobiopterin

in phenylketonuria patients with certain phenylalanine hydroxylase mutations.

Proc. Natl. Acad. Sci. U. S. A. 101:16903, 2004.

Gámez, A., Sarkissian, C.N., Wang, L., Kim, W., Straub, M., Patch, M.G., Chen,

L., Striepeke, S., Fitzpatrick, P., Lemontt, J.F., O’Neill, C., Scriver, C.R., Stevens,

R.C. Development of pegylated forms of recombinant Rhodosporidium toruloides

phenylalanine ammonia-lyase for the treatment of classical phenylketonuria. Mol.

Ther. 11:986, 2005.

Han, G.W., Schwarzenbacher, R., Page, R., et al. Crystal structure of an alanineglyoxylate

aminotransferase from Anabena sp at 1.70 Å resolution reveals a noncovalently

linked PLP cofactor. Proteins 58:971, 2005.

Levin, I., Miller, M.D., Schwarzenbacher, R., et al. Crystal structure of an indigoidine

synthase A (IndA)-like protein (TM1464) from Thermotoga maritima at 1.90 Å

resolution reveals a new fold. Proteins 59:864, 2005.

Matalon, R., Michals-Matalon, K., Koch, R., Grady, J., Tyring, S., Stevens, R.C.

Response of patients with phenylketonuria in the US to tetrahydrobiopterin. Mol.

Genet. Metab., in press.

Mathews, I., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of

S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) from Thermotoga

maritima at 2.0 Å resolution reveals a new fold. Proteins 59:869, 2005.

Page, R., Deacon, A.M., Lesley, S., Stevens, R.C. Shotgun crystallization strategy

for structural genomics, II: crystallization and conditions that produce high resolution

structures for T maritima proteins. J. Funct. Struct. Genomics 6:209, 2005.

Page, R., Peti, W., Wilson, I.A., Stevens, R.C., Wüthrich, K. NMR screening and

crystal quality of bacterially expressed prokaryotic and eukaryotic proteins in a

structural genomics pipeline. Proc. Natl. Acad. Sci. U. S. A. 102:1901, 2005.

Pérez, B., Desviat, L.R., Gomez-Puertas, P., Martinez, A., Stevens, R.C., Ugarte, M.

Kinetic and stability analysis of PKU mutations identified in BH4-responsive patients.

Mol .Genet. Metab., in press.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Peti, W., Johnson, M.A., Hermann, T., Newman, B.W., Buchmeier, M.J., Nelson, M.,

Joseph, J., Page, R., Stevens, R.C., Kuhn, P., Wüthrich, K. Structural genomics of

the severe acute respiratory syndrome coronavirus: nuclear magnetic resonance

structure of the protein nsP7. J. Virol . 79:12905, 2005.

Peti, W., Page, R., Wilson, I., Stevens, R., Wüthrich, K. Structural proteomics

pipeline miniaturized using micro expression and microcoil NMR. J. Struct. Funct.

Genomics, in press.

Pey, A.L., Pérez, B., Desviat, L.R., Martinez, M.A., Aguado, C., Erlandsen, H.,

Gámez, A., Stevens, R.C., Thorolfsson, M., Ugarte, M., Martinez, A. Mechanisms

underlying responsiveness to tetrahydrobiopterin in mild phenylketonuria mutations.

Hum. Mutat. 24:388, 2004.

Ricci, J.S., Stevens, R.C., McMullan, R.K., Klooster, W.T. The crystal structure of

strontium hydroxide octahydrate, Sr(OH)2.8H 2 O at 20, 100, and 200 K from neutron

diffraction. Acta Crystrallogr. B 61:381. 2005.

Rife, C., Schwarzenbacher, R., McMullen, D., et al. Crystal structure of a putative

modulator of DNA gyrase (pmbA) from Thermotoga maritima at 1.95 Å resolution

reveals a new fold. Proteins 61:444, 2005.

Rife, C., Schwarzenbacher, R., McMullen, D., et al. Crystal structure of a global

regulatory protein CsrA from Pseudomonas putida at 2.05 Å resolution reveals a

new fold. Proteins 61:449, 2005.

Saikatendu, K.S., Joseph, J.S., Subramanian, V., Clayton, T., Griffith, M., Moy,

K., Velasquez, J., Neuman, B.W., Buchmeier, M.J., Stevens, R.C., Kuhn, P.

Structural basis of severe acute respiratory syndrome coronavirus (SARS-CoV) ADPribose-1′′-phosphate

(Appr-1′′-p) dephosphorylation by a conserved domain of

nsP3. Structure, in press.

Scriver, C.R., Hurtubise, M., Prevost, L., Phommarinh, M., Konecki, D., Erlandsen,

H., Stevens, R.C., Waters, P.J., Ryan, S., McDonald, D., Sarkissan C. A PAH

gene knowledge base: content, informatics, utilization. In: PKU and BH 4 : Advances

in Phenylketonuria and Tetrahydrobiopterin Research. Blaue, N. (Ed.), SPS Publications,

Heilbrun, Germany, in press.

Swaminathan, S., Stevens, R.C. Three-dimensional protein structures of botulinum

neurotoxin light chains serotypes A, B, and E. In: Treatments from Toxins: The

Therapeutic Potential of Clostridial Neurotoxins. Foster, K., Hambleton, P., Shone,

C. (Eds.). CRC Press, Boca Raton, FL, in press.

Wang, L., Gámez, A., Sarkissian, C.N., Straub, M., Patch, M.G., Han, G.W.,

Striepeke, S., Fitzpatrick, P., Scriver, C.R., Stevens, R.C. Structure-based chemical

modification strategy for enzyme replacement treatment of phenylketonuria.

Mol. Genet. Metab. 86:134, 2005.

Xu, Q., Schwarzenbacher, R., McMullen, D., et al. Crystal structure of a formiminotetrahydrofolate

cyclodeaminase (TM1560) from Thermotoga maritima at 2.80 Å

resolution reveals a new fold. Proteins 58:976, 2005.

Yadav, M.K., Gerdts, C.J., Sanishvili, R., Smith, W., Roach, L.S., Ismagilov, R.F.,

Kuhn, P., Stevens, R.C. In situ data collection and structure refinement from

microcapillary protein crystallization. J. Appl. Crystallogr., in press.

Nuclear Magnetic Resonance

in Structural Biology and

Structural Genomics

K. Wüthrich, M. Almeida, L. Columbus, T. Etezady,

M. Geralt, S. Hiller, R. Horst, M. Johnson, W.J. Placzek,

W. Peti, P. Serrano

Members of our laboratory participate in the

Joint Center for Structural Genomics (JCSG),

the JCSG Center for Innovative Membrane


Protein Technologies, and the Functional and Structural

Proteomics Analysis of SARS-CoV–Related Proteins

Consortium. As part of these studies on protein structure,

we develop and use nuclear magnetic resonance

(NMR) methods to screen recombinant protein preparations

for folded proteins. We are also exploring the

use of microcoil NMR equipment combined with microexpression

of proteins. We also use NMR spectroscopy to

determine the structure of selected proteins from the

proteomes under study in the structural genomics programs.

Some of our research is described in the following

sections.

NMR SCREENING OF THERMOTOGA MARITIMA

MEMBRANE PROTEINS

A total of 45 predicted α-helical membrane proteins

from Thermotoga maritima were selected as potential

targets for solution NMR structural studies. These

proteins have between 1 and 4 predicted helical transmembrane

segments and have molecular weights less

than 16 kD. Of the 45 targets, 10 were overexpressed

in Escherichia coli, and 8 of these 10 localized to

the bacterial membrane. These 8 protein targets were

purified and screened to determine efficient detergents

for solubilization.

To evaluate the fold and the aggregation state of

the proteins in the best conditions thus identified, we

used 1-dimensional 1 H NMR spectroscopy to screen

the targets. For 3 of the 8 proteins, the NMR spectra

indicated soluble protein-detergent complexes. The

transverse relaxation optimized spectroscopy correlation

spectra of these 3 targets provided evidence that

these 3 proteins are folded helical proteins. Experiments

are under way for NMR assignment and structure determination

of these α-helical membrane proteins in mixed

micelles with detergents.

STRUCTURE DETERMINATIONS OF CONSERVED

HYPOTHETICAL PROTEINS FROM T MARITIMA

The NMR structure of the conserved hypothetical

protein TM1816 from T maritima has an α/β topology

with 3 α-helices and a 5-stranded β-sheet. The molecular

architecture of TM1816 is similar to that of 2 other

conserved hypothetical proteins, TM1290 from T maritima

(33% sequence identity) and MTH1175 from

Methanobacterium thermoautotrophicum (30% sequence

identity). These 3 proteins belong to the cluster of

orthologous groups 1433 and are structurally similar

to the Azobacter vinelandii iron, molybdenum cofactor-binding

protein NafY. TM1816 is unique among

the 3 homologs because it contains a histidine residue

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 177

corresponding to the one that is crucial for cofactor

binding in NafY.

TM0487 is a 104-residue protein from T maritima

that was identified via NMR screening as a potential

target for NMR structure determination. The 3-dimensional

structure of TM0487 provides a foundation for

functional studies of an entire class of proteins, because

TM0487 has a large number of homologs on the amino

acid sequence level, including 216 nonredundant

sequences that contain a type 59 domain of unknown

function. So far, a 3-dimensional structure has not been

determined for any of these homologous proteins. The

conserved residues among the aforementioned 216

sequences are clustered in the hydrophobic core of the

TM0487 fold and in a putative active site exposed to

the solvent. Overall, strong evidence indicates that the

TM0487 fold is preserved in all of this class of domains

of unknown function, so that this structure determination

provides a foundation for focused functional studies

of a wide variety of otherwise so far only minimally

characterized proteins.

NMR STUDIES OF AN ACYL CARRIER PROTEIN

FROM THE CYANOBACTERIUM ANABAENA

Asl1650, a protein obtained from the cyanobacterium

Anabaena, was identified as an ortholog of a

mouse protein domain as part of a JCSG bioinformatics

strategy to extend information on the protein folding

space of eukaryotic proteins. The protein was

selected for NMR structure determination on the basis

of an NMR screen of recombinant mouse protein homologs

expressed in E coli.

Acyl carrier proteins (ACPs) are central components

of complex multienzyme systems that function in the

metabolism of all living organisms. These systems catalyze

the biosynthesis of fatty acids, signaling molecules,

and bioactive natural products. The polyketide

synthases and nonribosomal peptide synthetases of

microorganisms produce compounds with antibiotic

and anticancer activities. An understanding of structure-function

relationships in these widely distributed

enzyme systems is thus of obvious interest for the design

of new therapeutic compounds.

The protein Asl1650 is only distantly related to

previously characterized ACPs. It was derived from

Anabaena sp PCC 7120, a filamentous cyanobacterium.

Members of this genus of cyanobacteria produce

a variety of bioactive compounds, which are as

yet only poorly characterized. We determined the solution

structure of Asl1650 by using high-resolution NMR


178 MOLECULAR BIOLOGY 2005

spectroscopy. The structure had a surprising similarity

to the structures of peptidyl carrier protein domains,

which usually occur as single domains of giant, multifunctional

proteins. A variant active-site sequence,

asparagine–serine–serine, occurs in similar orientation

to the aspartic acid–serine–leucine sequence of known

ACPs. These structural similarities suggest that Asl1650

may function as a discrete peptidyl carrier protein

domain in a nonribosomal peptide synthetase pathway

or a hybrid polyketide synthase–nonribosomal peptide

synthetase pathway.

PUBLICATIONS

Almeida, M.S., Peti, W., Wüthrich, K. 1 H-, 13 C- and 15 N-NMR assignment of the

conserved hypothetical protein TM0487 from Thermotoga maritima. J. Biomol.

NMR 29:453, 2004.

Etezady-Esfarjani, T., Herrmann, T., Peti, W., Klock, H.E., Lesley, S.A., Wüthrich, K.

NMR structure determination of the hypothetical protein TM1290 from Thermotoga

maritima using automated NOESY analysis. J. Biomol. NMR 29:403, 2004.

Page, R., Peti, W., Wilson, I.A., Stevens, R.C., Wüthrich, K. NMR screening and

crystal quality of bacterially expressed prokaryotic and eukaryotic proteins in a

structural genomics pipeline. Proc. Natl. Acad. Sci. U. S. A. 102:1901, 2005.

Peti, W., Etezady-Esfarjani, T., Herrmann, T., Klock, H.E., Lesley, S.A., Wüthrich, K.

NMR for structural proteomics of Thermotoga maritima: screening and structure

determination. J. Struct. Funct. Genomics 5:205, 2004.

Peti, W., Norcross, J., Eldridge, G., O’Neil-Johnson, M. Biomolecular NMR using

a microcoil NMR probe: new technique for the chemical shift assignment of aromatic

side chains in proteins. J. Am. Chem. Soc. 126:5873, 2004.

Nuclear Magnetic Resonance of

3-Dimensional Structure and

Dynamics of Proteins in Solution

P.E. Wright, H.J. Dyson, R. Burge, R. De Guzman,

T. Dunzendorfer-Matt, J. Ferreon, N. Greenman,

T.-H. Huang, M. Kostic, J. Lansing, B. Lee, M. Landes,

M. Martinez-Yamout, T. Nishikawa, J. Wojciak, M. Zeeb,

E. Manlapaz, L.L. Tennant, J. Chung, D.A. Case,

J. Gottesfeld, R. Evans,* M. Montminy*

* Salk Institute, La Jolla, California

We use multidimensional nuclear magnetic

resonance (NMR) spectroscopy to investigate

the structures, dynamics, and interactions

of proteins in solution. Such studies are essential

for understanding the mechanisms of action of these

proteins and for elucidating structure-function relationships.

The focus of our current research is protein-protein

and protein–nucleic acid interactions involved in

the regulation of gene expression.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

TRANSCRIPTION FACTOR–NUCLEIC ACID COMPLEXES

NMR methods are being used to determine the

3-dimensional structures and intramolecular dynamics

of zinc finger motifs from several eukaryotic transcriptional

regulatory proteins, both free and complexed with

target nucleic acid. Zinc fingers are among the most

abundant domains in eukaryotic genomes. They play a

central role in the regulation of gene expression at both

the transcriptional and the posttranscriptional levels,

mediated through their interactions with DNA, RNA,

or protein components of the transcriptional machinery.

The C 2 H 2 zinc finger, first identified in transcription

factor IIIA (TFIIIA), is used by numerous transcription

factors to achieve sequence-specific recognition of DNA.

There is growing evidence, however, that some C 2 H 2

zinc finger proteins control gene expression both through

their interactions with DNA regulatory elements and,

at the posttranscriptional level, by binding to RNA.

The best-characterized example of a C 2 H 2 zinc

finger protein that binds specifically to both DNA and

to RNA is TFIIIA, which contains 9 zinc fingers. We

showed previously that different subsets of zinc fingers

are responsible for high-affinity binding of TFIIIA to DNA

(fingers 1–3) and to 5S RNA (fingers 4–6). To obtain

insights into the mechanism by which the TFIIIA zinc

fingers recognize both DNA and RNA, we are using NMR

methods to determine the structures of the complex

formed by zf1-3 (a protein containing fingers 1–3) with

DNA and by zf4-6 (a protein consisting of fingers 4–6)

with a fragment of 5S RNA.

Three-dimensional structures were determined previously

for the complex of zf1-3 with the cognate 15-bp

oligonucleotide duplex. The structures contain several

novel features and reveal that prevailing models of DNA

recognition, which assume that zinc fingers are independent

modules that contact bases through a limited

set of amino acids, are outmoded.

In addition to its role in binding to and regulating

the 5S RNA gene, TFIIIA also forms a complex with the

5S RNA transcript. We recently determined the NMR

structure of the complex formed by zinc fingers 4–6 with

a truncated form of 5S RNA (Fig. 1). The structure has

provided important insights into the structural basis for

5S RNA recognition. Finger 4 of the protein recognizes

both the structure of the RNA backbone and the specific

bases in the loop E motif of the RNA, in a classic lockand-key

interaction. Fingers 5 and 6, with a single residue

between them, undergo mutual induced-fit folding

with the loop A region of the RNA, which is highly flexible

in the absence of the protein.


Fig. 1. Structure of zinc fingers 4–6 of TFIIIA bound to 5S RNA.

The protein backbone is shown as a ribbon, and the phosphate

backbone and bases of the RNA are displayed as gray tubes.

NMR studies of 2 alternate splice variants of the

Wilms tumor zinc finger protein are in progress. These

proteins differ only through insertion of 3 additional

amino acids (the tripeptide lysine-threonine-serine) in

the linker between fingers 3 and 4, yet have marked

differences in their DNA-binding properties and subcellular

localization. 15 N relaxation measurements indicate

that the insertion increases the flexibility of the

linker between fingers 3 and 4 and abrogates binding

of the fourth zinc finger to its cognate site in the DNA

major groove, thereby modulating DNA-binding activity.

The x-ray structure of the DNA complex has now been

determined, and NMR studies of RNA binding are in

progress. We have also determined the structure of the

first member of a novel class of C 2 H 2 zinc finger proteins

that bind specifically to double-stranded RNA.

Several novel zinc binding motifs have recently

been identified that mediate gene expression at the

posttranscriptional level by regulating mRNA processing

and metabolism. Regulatory proteins of the TIS11

family bind specifically, through a pair of novel CCCH

zinc fingers, to the adenosine-uridine–rich element in

the 3′ untranslated region of short-lived cytokine, growth

factor, and protooncogene mRNAs and control expression

by promoting rapid degradation of the message. We

recently determined the NMR structure of the complex

formed between the tandem zinc finger domain of TIS11d

and its binding site on the adenosine-uridine–rich ele-

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 179

ment. This structure showed sequence-specific recognition

of single-stranded RNA through formation of a

network of hydrogen bonds between the polypeptide

backbone and the Watson-Crick edges of the bases.

PROTEIN-PROTEIN INTERACTIONS IN

TRANSCRIPTIONAL REGULATION

Transcriptional regulation in eukaryotes relies on

protein-protein interactions between DNA-bound factors

and coactivators that, in turn, interact with the basal

transcription machinery. The transcriptional coactivator

CREB-binding protein (CBP) and its homolog p300 play

an essential role in cell growth, differentiation, and

development. Understanding the molecular mechanisms

by which CBP and p300 recognize their various target

proteins is of fundamental biomedical importance. CBP

and p300 have been implicated in diseases such as

leukemia, cancer, and mental retardation and are novel

targets for therapeutic intervention.

We previously determined the structure of the kinaseinducible

activation domain of the transcription factor

CREB bound to its target domain (the KIX domain) in

CBP. Ongoing work is directed toward mapping the interactions

between KIX and the transcriptional activation

domains of the proto-oncogene c-Myb and of the mixedlineage

leukemia protein. The solution structure of the

ternary complex composed of KIX, c-Myb, and the mixedlineage

leukemia protein has been completed (Fig. 2)

and provides insights into the structural basis for the

ability of the KIX domain to interact simultaneously and

allosterically with 2 different effectors. Our work has also

provided new understanding of the thermodynamics of

the coupled folding and binding processes involved in

interaction of KIX with transcriptional activation domains.

Fig. 2. Structure of the ternary complex between the KIX domain

of CBP (pale gray) and the transcriptional activation domains of

c-Myb and the mixed-lineage leukemia protein (MLL).


180 MOLECULAR BIOLOGY 2005

Recently, we determined the structure of the complex

between the hypoxia-inducible factor Hif-1α and

the CH1 domain of CBP. The interaction between Hif-1α

and CBP/p300 is of major therapeutic interest because

of the central role Hif-1α plays in tumor progression and

metastasis; disruption of this interaction leads to attenuation

of tumor growth. A protein named CITED2 functions

as a negative feedback regulator of the hypoxic

response by competing with Hif-1α for binding to the

CH1 domain of CBP. We determined the structure of

the complex formed between CITED2 and the CH1

domain and were able to show that the CH1 domain

is folded into a stable 3-dimensional structure even in

the absence of binding partners. The intrinsically unstructured

Hif-1α and CITED2 domains use partly overlapping

surfaces of the CH1 motif to achieve high-affinity

binding and compete effectively with each other for

CBP/p300. The structure of another zinc-binding module

of CBP, the ZZ domain, has a novel fold (Fig. 3),

but its function is not yet understood. We are continuing

to map the multiplicity of interactions between CBP/p300

domains and their numerous biological targets to understand

the complex interplay of interactions that mediate

key biological processes in health and disease.

Fig. 3. Structure of the ZZ zinc finger domain of CBP.

PUBLICATIONS

De Guzman, R.N., Goto, N.K., Dyson, H.J., Wright, P.E. Structural basis for cooperative

transcription factor binding to the CBP coactivator. J. Mol. Biol., in press.

De Guzman, R.N., Wojciak, J.M., Martinez-Yamout, M.A., Dyson, H.J., Wright,

P.E. CBP/p300 TAZ1 domain forms a structural scaffold for ligand binding. Biochemistry

44:490, 2005.

Dyson, H.J., Wright, P.E. Intrinsically unstructured proteins and their function. Nat.

Rev. Mol. Cell Biol. 6:197, 2005.

Gearhart, M.D., Dickinson, L., Ehley, J., Melander, C., Dervan, P.B., Wright, P.E., Gottesfeld,

J.M. Inhibition of DNA binding by human estrogen related receptor-2 and estrogen

receptor α with minor groove binding polyamides. Biochemistry 44:4196, 2005.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Legge, G.B., Martinez-Yamout, M.A., Hambly, D.M., Trinh, T., Lee, B.M., Dyson,

H.J., Wright, P.E. ZZ domain of CBP: an unusual zinc finger fold in a protein interaction

module. J. Mol. Biol. 343:1081, 2004.

Möller, H.M., Martinez-Yamout, M.A., Dyson, H.J. Wright, P.E. Solution structure

of the N-terminal zinc fingers of the Xenopus laevis double-stranded RNA-binding

protein ZFa. J. Mol. Biol. 351:718, 2005.

Folding of Proteins and

Protein Fragments

P.E. Wright, H.J. Dyson, C. Nishimura, D. Felitsky, Y. Yao,

J. Chung, L.L. Tennant, V. Bychkova*

* Institute of Protein Research, Puschino, Russia

The molecular mechanism by which proteins fold

into their 3-dimensional structures remains one

of the most important unsolved problems in structural

biology. Nuclear magnetic resonance (NMR) spectroscopy

is uniquely suited to provide information on

the structure of transient intermediates formed during

protein folding. Previously, we used NMR methods to

show that many peptide fragments of proteins have a

tendency to adopt folded conformations in water solution.

The presence of transiently populated folded structures,

including reverse turns, helices, nascent helices,

and hydrophobic clusters, in water solutions of short

peptides has important implications for initiation of protein

folding. Formation of elements of secondary structure

probably plays an important role in the initiation

of protein folding by reducing the number of conformations

that must be explored by the polypeptide chain and

by directing subsequent folding pathways.

APOMYOGLOBIN FOLDING PATHWAY

A major program in our laboratory is directed

toward a structural and mechanistic description of the

apomyoglobin folding pathway. Previously, we used

quenched-flow pulse labeling methods in conjunction

with 2-dimensional NMR spectroscopy to map the

kinetic folding pathway of the wild-type protein. With

these methods, we showed that an intermediate in

which the A, G, and H helices adopt hydrogen-bonded

secondary structure is formed within 6 ms of the initiation

of refolding. Folding then proceeds by stabilization

of structure in the B helix and then in the C and

E helices. We are using carefully selected myoglobin

mutants and both optical stopped-flow spectroscopy

and NMR methods to further probe the kinetic folding

pathway. For some of the mutants studied, the changes

in amino acid sequence resulted in changes in the fold-


ing pathway of the protein. These experiments are providing

novel insights into both the local and the longrange

interactions that stabilize the kinetic folding

intermediate. Of particular importance, long-range

interactions have been observed that indicate nativelike

packing of some of the helices in the kinetic molten

globule intermediate.

Apomyoglobin provides a unique opportunity for

detailed characterization of the structure and dynamics

of a protein-folding intermediate. Conditions were previously

identified under which the apomyoglobin molten

globule intermediate is sufficiently stable for acquisition

of multidimensional heteronuclear NMR spectra.

Analysis of 13 C and other chemical shifts and measurements

of polypeptide dynamics provided unprecedented

insights into the structure of this state.

The A, G, and H helices and part of the B helix are

folded and form the core of the molten globule. This

core is stabilized by relatively nonspecific hydrophobic

interactions that restrict the motions of the polypeptide

chain. Fluctuating helical structure is formed in regions

outside the core, although the population of helix is low

and the chain retains considerable flexibility. The F helix

acts as a gate for heme binding and only adopts stable

structure in the fully folded holoprotein.

The acid-denatured (unfolded) state of apomyoglobin

is an excellent model for the fluctuating local interactions

that lead to the transient formation of unstable

elements of secondary structure and local hydrophobic

clusters during the earliest stages of folding. NMR data

indicated substantial formation of helical secondary

structure in the acid-denatured state in regions that form

the A and H helices in the folded protein and also

revealed nonnative structure in the D and E helix region.

Because the A and H regions adopt stabilized helical

structure in the earliest detectable folding intermediate,

these results lend strong support to folding models

in which spontaneous formation of local elements of

secondary structure plays a role in initiating formation

of the A-[B]-G-H molten globule folding intermediate.

In addition to formation of transient helical structure,

formation of local hydrophobic clusters has been detected

by using 15 N relaxation measurements. Significantly,

these clusters are formed in regions where the average

surface area buried upon folding is large. In contrast

to acid-denatured unfolded apomyoglobin, the ureadenatured

state is largely devoid of structure, although

residual hydrophobic interactions have been detected

by using relaxation measurements.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 181

We measured residual dipolar couplings for unfolded

states of apomyoglobin by using partial alignment in

strained polyacrylamide gels. These data provide novel

insights into the structure and dynamics of the unfolded

polypeptide chain. We have shown that the residual

dipolar couplings arise from the well-known statistical

properties of flexible polypeptide chains. Residual dipolar

couplings provide valuable insights into the dynamic

and conformational propensities of unfolded and partly

folded states of proteins and hold great promise for

charting the upper reaches of protein-folding landscapes.

To probe long-range interactions in unfolded and

partially folded states of apomyoglobin, we introduced

spin-label probes at several sites throughout the polypeptide

chain. These experiments led to the surprising

discovery that structures with nativelike topology exist

within the ensemble of conformations formed by the

acid-denatured state of apomyoglobin. They also indicated

that the packing of helices in the molten globule

state is similar to that in the native folded protein.

The view of protein folding that results from our

work on apomyoglobin is one in which collapse of the

polypeptide chain to form increasingly compact states

leads to progressive accumulation of secondary structure

and increasing restriction of fluctuations in the

polypeptide backbone. Chain flexibility is greatest at

the earliest stages of folding, in which transient elements

of secondary structure and local hydrophobic

clusters are formed. As the folding protein becomes

increasingly compact, backbone motions become more

restricted, the hydrophobic core is formed and extended,

and nascent elements of secondary structure are progressively

stabilized. The ordered tertiary structure

characteristic of the native protein, with well-packed

side chains and relatively low-amplitude local dynamics,

appears to form rather late in folding.

We recently introduced a variation on the classic

quench-flow technique, which makes use of the capabilities

of modern NMR spectrometers and heteronuclear

NMR experiments, to study the proteins labeled

along the folding pathway in an unfolded state in an

aprotic organic solvent. This method allows detection

of many more amide proton probes than in the classic

method, which required formation of the fully folded

protein and the measurement of the protein’s NMR

spectrum in water solutions (Fig. 1). This method is

particularly useful in documenting changes in the folding

pathway that result in the destabilization of parts of the

protein in the molten globule intermediate. We recently


182 MOLECULAR BIOLOGY 2005

Fig. 1. High-resolution view of the backbone structure of the

6.4-ms burst-phase kinetic folding intermediate of apomyoglobin.

The tube thickness and darkness indicate the extent of folding into

helical structure. Helices that are fully folded are indicated by thick,

dark tubes. Regions that are partly folded are intermediate in thickness

and shade, and regions of the protein that remain fully unstructured

in the kinetic intermediate are represented by thin lines.

introduced self-compensating mutations designed to

change the amino acid sequence such that the average

area buried upon folding in the A and E helix regions is

significantly changed, while the 3-dimensional structure

of the final folded state remains the same. These studies

indicated that the average area buried upon folding is an

accurate predictor of those parts of the apomyoglobin

molecule that will fold first and participate in the molten

globule intermediate (Fig. 2).

FOLDING-UNFOLDING TRANSITIONS IN CELLULAR

METABOLISM

Many species of bacteria sense and respond to their

own population density by an intricate autoregulatory

mechanism known as quorum sensing; the bacteria

release extracellular signal molecules, called autoinducers,

for cell-cell communication within and between

bacterial species. A number of bacteria appear to use

quorum sensing for regulation of gene expression in

response to fluctuations in cell population density. Processes

regulated in this way include symbiosis, virulence,

competence, conjugation, production of antibiotics, motility,

sporulation, and formation of biofilms.

We determined the 3-dimensional solution structure

of a complex composed of the N-terminal 171 residues

of the quorum-sensing protein SdiA of Escherichia coli

and an autoinducer molecule, N-octanoyl-1-homoserine

lactone (HSL). The SdiA-HSL system shows the

“folding switch” behavior associated with quorum-sensing

factors produced by other bacterial species. In the

presence of HSL, the SdiA protein is stable and folded

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Fig. 2. Correlation between average surface area buried upon

folding (AABUF, gray line) and regions of apomyoglobin that are

folded in the kinetic burst-phase intermediate. Folded regions are

indicated by high values of the proton occupancy (A0, black circles).

Data are shown for the wild-type protein (A) and for a mutant protein

(B) in which hydrophobic residues are moved from the A helix

into the E helix region, thereby changing the folding pathway in a

predictable manner.

and can be produced in good yields from an E coli

expression system. In the absence of the autoinducer,

the protein is expressed into inclusion bodies. Samples

of the SdiA-HSL complex can be denatured but

cannot be refolded in aqueous buffers. The solution

structure of the complex provides a likely explanation

for this behavior. The autoinducer molecule is tightly

bound in a deep pocket in the hydrophobic core and

is bounded by specific hydrogen bonds to the side

chains of conserved residues. The autoinducer thus

forms an integral part of the hydrophobic core of the

folded SdiA.

PUBLICATIONS

Dyson, H.J., Wright, P.E. Elucidation of the protein folding landscape by NMR.

Methods Enzymol. 394:299, 2005.

Dyson, H.J., Wright, P.E. Intrinsically unstructured proteins and their functions.

Nat. Rev. Mol. Cell Biol. 6:197, 2005.

Nishimura, C., Dyson, H.J., Wright, P.E. Enhanced picture of protein-folding intermediates

using organic solvents in H/D exchange and quench-flow experiments.

Proc. Natl Acad. Sci. U. S. A. 102:4765, 2005.

Nishimura, C., Dyson, H.J. Wright, P.E. Identification of native and nonnative

structure in kinetic folding intermediates of apomyoglobin. J. Mol. Biol., in press.

Nishimura, C., Lietzow, M.A., Dyson H.J., Wright, P.E. Sequence determinants of

a protein folding pathway. J. Mol. Biol. 351:383, 2005.


Nuclear Magnetic Resonance

Studies of the Structure and

Dynamics of Enzymes

H.J. Dyson, P.E. Wright, D. Boehr, M.O. Ebert, G. Kroon,

J. Lansing, C.W. Lee, M. Martinez-Yamout, D. McElheny,

N.E. Preece, K. Sugase, H.S. Won, Y. Yao, L.L. Tennant,

J. Chung, C.L. Brooks, S.J. Benkovic,* A. Holmgren**

* Pennsylvania State University, University Park, Pennsylvania

** Karolinska Institutet, Stockholm, Sweden

We use site-specific information from nuclear

magnetic resonance (NMR) to further the

understanding of enzyme function through

study of enzyme structure and dynamics. We focus on

the mechanisms of enzymes and the relationship

between dynamics and function in cellular control by

thiol-disulfide chemistry.

DYNAMICS IN ENZYME ACTION

Dynamic processes are implicit in the catalytic function

of all enzymes. We use state-of-the-art NMR methods

to elucidate the dynamic properties of several enzymes.

New methods have been developed for analysis of NMR

relaxation data for proteins that tumble anisotropically

and for analysis of slow time scale motions.

Dihydrofolate reductase plays a central role in folate

metabolism and is the target enzyme for a number of

anticancer agents. 15 N relaxation experiments on dihydrofolate

reductase from Escherichia coli revealed a

rich diversity of backbone dynamics for a broad range

of time scales (picoseconds to milliseconds). These studies

were extended to additional intermediates in the

reaction cycle and to forms of the enzyme with mutations

at various motional “hot spots.”

In addition, we are using 2 H relaxation measurements

in triple-labeled dihydrofolate reductase to elucidate

the dynamics of critical active-site side chains.

So far, we have identified functionally important motions

in loops that control access to the active site of the

reductase on the same time scale as the hydride transfer

chemistry. These motions become attenuated once the

NADPH cofactor is bound in the active site, locking the

nicotinamide ring in a geometry conducive to hydride

transfer to substrate. We also found evidence of motion

of active-site side chains that are implicated in the

catalytic process.

Most recently, we used relaxation dispersion measurements

to obtain direct information on microsecond-

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 183

millisecond time scale motions in dihydrofolate reductase,

allowing us to characterize the structures of the

excited states involved in some of these catalysis-relevant

processes. Fluctuations between these states, which

involve motions of the nicotinamide ring of the cofactor

into and out of the active site, occur on a time scale

that is directly relevant to the structural transitions

involved in progression through the catalytic cycle.

Dihydrofolate reductase is also the test system for

a series of experiments to address the question, If all

of the chemistry goes on at the active site, what is the

purpose of the rest of the enzyme? We will use a series

of chimeric mutants, synthesized by our collaborator

S.J. Benkovic, Pennsylvania State University, by using

a library approach. The purpose of these experiments

is to test the hypothesis that local variations in amino

acid sequence, 3-dimensional structure, and polypeptide

chain dynamics strongly influence the local interactions

that mediate enzyme catalysis and may constitute the

essential circumstance that allows enzymes to achieve

high turnover rates as well as exquisite specificity in

their reactions. A combination of NMR structure and

dynamics measurements, single-molecule fluorescence

measurements, and analysis of the catalytic steps in

these mutant proteins will provide new insights into

the role of the protein in enzyme catalysis.

REDOX CONTROL BY THIOL-DISULFIDE CHEMISTRY

Many cellular functions are regulated by thiol-disulfide

chemistry. The importance of redox chemistry, particularly

disulfide-dithiol equilibria, in cellular control

mechanisms has only recently been recognized. For

example, the chaperone heat-shock protein 33 (Hsp33)

is regulated by a redox switch; the C-terminal domain

of Hsp33 contains cysteines that are reduced and bound

to zinc under normoxic conditions, but upon oxidation,

the zinc is lost and disulfide bonds form. Interestingly,

the zinc-bound form of the C-terminal domain is well

structured, with a distinctive fold. NMR studies revealed

that upon oxidation, the C-terminal domain becomes

unstructured. We think that this loss of local structure

exposes a dimerization site. Thus, under oxidative stress

conditions, the chaperone dimerizes to the active form.

We did an extensive study of the structural basis

for the activity of several thiol-disulfide enzymes. Thioredoxin,

a small, 108-residue thiol-disulfide oxidoreductase,

has many functions in the cell, including reduction

of ribonucleotides to form deoxyribonucleotides for DNA

synthesis. A primary function of thioredoxin in the cell

is as a protein disulfide reductase, a function vital for


184 MOLECULAR BIOLOGY 2005

the prevention of misfolded proteins in vivo. The E coli

thioredoxin system has been fully characterized by using

NMR, including the calculation of high-resolution structures

for both the oxidized (disulfide) and the reduced

(dithiol) forms of the protein.

Using backbone dynamics and amide proton hydrogen

exchange, we found that functional differences in

phage systems between oxidized and reduced thioredoxin

were due to differences in the flexibility of the molecules

rather than to structural differences. We also delineated

the mechanism of E coli thioredoxin. We found that the

reduction reaction of thioredoxin depends critically on

the movement of protons, during the 2-electron–2-proton

transfer reaction, as a substrate disulfide is reduced.

We are investigating a variant E coli thioredoxin with

an N-terminal extension that binds zinc. This exciting

new molecule may be another example of a redox-active,

zinc-binding protein, previously exemplified by the redoxswitch

domain of the chaperone Hsp33.

Glutaredoxins are another major class of thiol-disulfide

regulatory proteins. We recently determined the

structure of glutaredoxin-2 from E coli. This protein

appears to be a link between the glutaredoxin-thioredoxin

class of small thiol-active proteins and the extensive

glutathione-S-transferase class of detoxification enzymes.

Glutaredoxins are thought to be involved in the processes

that result in the attachment and removal of glutathione

and nitrosyl groups from redox-active proteins.

These processes, together with the formation of disulfide

bonds, regulate the activity of redox-active proteins

such as the transcription factor OxyR, which we

also study.

PUBLICATIONS

Chen, J., Won, H.-S., Im, W., Dyson, H.J., Brooks, C.L. III. Generation of nativelike

protein structures from limited NMR data, modern force fields and advanced

conformational sampling. J. Biomol. NMR 31:59, 2005.

McElheny, D., Schnell, J.R., Lansing, J.C., Dyson, H.J., Wright, P.E. Defining the

role of active-site loop fluctuations in dihydrofolate reductase catalysis. Proc. Natl.

Acad. Sci. U. S. A. 102:5032, 2005.

Venkitakrishnan, R.P., Zaborowski, E., McElheny, D., Benkovic, S.J., Dyson, H.J.,

Wright, P.E. Conformational changes in the active site loops of dihydrofolate reductase

during the catalytic cycle. Biochemistry 43:16046, 2004.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Ring Assemblies Mediating

ATP-Dependent Protein Folding

and Unfolding

A.L. Horwich, W.A. Fenton, E. Chapman, E. Koculi

Large ring assemblies function in many cellular

contexts as compartments within a compartment,

where actions can be carried out on a substrate

bound in the central space inside an oligomeric ring

by a high local concentration of surrounding active sites.

Both protein folding and unfolding are carried out in

an ATP-dependent fashion by such assemblies. We are

studying the essential double-ring components, chaperonins,

that assist protein folding to the native state.

We are focusing on the bacterial chaperonin GroEL

and more recently have been examining an opposite

number, an “unfoldase,” the bacterial heat-shock protein

100 ring assembly known as ClpA. In the past

year, we focused on polypeptide binding and ATPmediated

action by both machines, showing quite different

mechanisms.

GROEL-MEDIATED FOLDING

We are investigating polypeptide binding by an open

ring of GroEL that is mediated through contacts between

the exposed hydrophobic surface of nonnative polypeptide

and a hydrophobic lining of the open ring. This step

is one that potentially mediates unfolding of kinetically

trapped states. In collaboration with K. Wüthrich, Department

of Molecular Biology, using solution nuclear magnetic

resonance and transverse relaxation optimized

spectroscopy, we examined the structure of isotopelabeled

human dihydrofolate reductase bound to GroEL.

The resonances detected indicate that the reductase

does not occupy a stable tertiary structure while bound

to an open GroEL ring and also suggest that the enzyme

is undergoing conformational exchange. This unfolded

state was, however, productive; upon addition of ATP

and the cochaperonin GroES, a nativelike pattern of

resonances was recovered.

The binding of ATP and GroES triggers productive

GroEL-GroES–mediated folding in the encapsulated

now-hydrophilic cavity of the GroES-bound ring (Fig. 1).

By contrast, addition of ADP and GroES does not trigger

folding. Surprisingly, however, x-ray and solution

electron cryomicroscopy structures of GroEL-GroES-

ADP and GroEL–GroES–ADP–aluminum fluoride, which

is a folding-active state, are isomorphous. We noted


Fig. 1. Protein folding and unfolding by chaperone ring assemblies.

In protein folding mediated by the chaperonin GroEL (left), the energy

of binding ATP and the cochaperonin GroES are used to produce

rigid body movements of a GroEL ring that eject a bound nonnative

substrate polypeptide into a GroES-encapsulated central cavity,

switched from hydrophobic (shaded) to hydrophilic wall character,

where productive folding proceeds. The free energy provided by a

set of hydrogen bonds formed between the γ-phosphate of ATP and

the nucleotide pocket is critical to producing a power stroke of apical

domain movement that can eject the substrate polypeptide into

the folding chamber. In contrast, in ClpA-mediated unfolding (right),

this chaperone seems to use ATP hydrolysis by its D2 ATPase domain

to drive a forceful distalward movement of a loop facing its central

channel, exerting mechanical force on a bound protein that is proposed

to exert an unfolding action.

that these structure determinations were all carried out

in the absence of substrate polypeptide and that a

bound substrate potentially represents a load on the

ring to which it is bound, resisting nucleotide/GroESdriven

elevation and twist of the apical domain that are

associated with ejection of a bound polypeptide off

the cavity wall into the GroES-encapsulated cavity where

productive folding occurs. Thus, the γ-phosphate of ATP

might be critical to exerting a power stroke of apical

movement. Consistent with such an idea, we found that

addition of aluminum fluoride to a GroEL-GroES-ADPpolypeptide

complex triggered productive folding. Further,

we found that a substantial amount of free energy

was released upon binding of aluminum fluoride to

GroEL-GroES-ADP.

To directly monitor apical movement, we used fluorescence

resonance energy transfer between a fluorophore

placed on the stable equatorial base of a subunit

and a fluorophore placed in the apical domain (at a

position that moves ~30 Å during the transition of a

ring from unbound to GroES bound). Indeed, when no

substrate was present, the apical domains opened rapidly

(40 seconds).

Additional studies with fluorophores placed on GroEL

and GroES indicated that GroES can associate rapidly

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

with GroEL-polypeptide complexes in ADP, evidently

forming a collision complex, but subsequent apical

movement is impaired. We are using electron microscopy

to examine the putative collision state, because

it most likely is a state that is transiently populated in

the physiologic nucleotide ATP.

CLPA-MEDIATED UNFOLDING

MOLECULAR BIOLOGY 2005 185

ClpA recognizes terminal peptide tags of proteins

that are concordantly unfolded and translocated through

its central channel. The polypeptide is generally directly

translocated into a double-ring proteasome-like protease,

ClpP, where it is degraded. During this past year,

we used chemical cross-linkers placed on tag elements

to identify channel-facing structures of ClpA that bind

the tags and then did mutational analysis of the identified

regions. For example, the C-terminal 11-residue

ssrA peptide, which is added to proteins stalled at the

ribosome to recruit these chains to ClpA, binds to 3

loops in the central channel of ClpA, 2 at the level of

the proximal D1 ATPase domain and 1 at the level of

the distal D2 ATPase (Fig. 1). Interestingly, a mutation

at the point of insertion of the D2 loop into the channel

wall allows substrate binding but blocks unfolding/

translocation, suggesting that this loop, connected to

the more active D2 ATPase of ClpA, is a translocator

that pulls on bound polypeptide in association with

ATP hydrolysis, exerting a mechanical force that mediates

unfolding. Consistently, x-ray studies of different

nucleotide states have shown that 2 other such ring

components that act on nucleic acids, the phi12 packaging

motor and simian virus 40 T antigen, undergo

such movements of channel-facing loops.

PUBLICATIONS

Hinnerwisch, J., Fenton, W.A., Furtak, K., Farr, G.W., Horwich, A.L. Loops in the

central channel of ClpA chaperone mediate protein binding, unfolding, and translocation.

Cell 121:1029, 2005.

Horst, R., Bertelsen, E.B., Fiaux, J., Wider, G., Horwich, A.L., Wüthrich, K.

Direct NMR observation of a substrate protein bound to the chaperonin GroEL.

Proc. Natl. Acad. Sci. U. S. A. 102:000, 2005.

Motojima, F., Chaudhry, C., Fenton, W.A., Farr, G.W., Horwich, A.L. Substrate

polypeptide presents a load on the apical domains of the chaperonin GroEL. Proc.

Natl. Acad. Sci. U. S. A. 101:15005, 2004.


186 MOLECULAR BIOLOGY 2005

Chemical Regulation of

Gene Expression

J.M. Gottesfeld, D. Alvarez-Carbonell, R. Burnett, J. Chou,

D. Herman, K. Jennsen, S. Ku, P.B. Dervan*, K. Luger**

* California Institute of Technology, Pasadena, California

** Colorado State University, Fort Collins, Colorado

TRANSCRIPTION REGULATION WITH SMALL

MOLECULES

Pyrrole-imidazole polyamides are the only available

class of synthetic small molecules that can be

designed to bind predetermined DNA sequences

with affinities comparable to those of cellular gene regulatory

proteins. In collaboration with P.B. Dervan and

colleagues at the California Institute of Technology, we

showed that polyamides inhibit the DNA-binding activities

of various transcriptional regulatory proteins and

can be used to inhibit transcription in cell culture experiments.

Previous studies established that transcription

can be inhibited with polyamides by targeting the binding

sites for essential transcription regulatory proteins in

gene promoters in the cell nucleus. We also found that

site-specific DNA alkylation by polyamide-chlorambucil

conjugates within a coding region of a gene strongly

blocks transcription elongation by mammalian RNA

polymerase II, both in vitro and in reporter gene transfection

experiments in cell culture.

We screened a series of polyamide-chlorambucil

conjugates with different DNA sequence specificities

for effects on morphology and growth characteristics

of human colon carcinoma cell lines. We identified a

compound that causes cells to arrest in the G 2 /M stage

of the cell cycle, without any apparent cytotoxic effects.

This change in growth properties required both the DNAbinding

specificity of the polyamide and the alkylator

moiety, suggesting that growth arrest is due to the silencing

of a set of specific genes by site-specific alkylation.

Surprisingly, DNA microarray analysis indicated that

only a few genes of about 18,000 genes probed were

significantly downregulated by this polyamide, and

reverse transcriptase–polymerase chain reaction and

Western blotting experiments confirmed that among

these genes, a member of the human gene family that

encodes histone H4, an essential component of chromatin,

is significantly downregulated. This particular

gene, the gene for histone H4c, is actively transcribed

in various cancer cell lines but is only moderately

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

transcribed in normal cells and tissues. Downregulation

of H4c mRNA by small interfering RNA yielded the

same cellular response, providing target validation.

The gene for histone H4c contains binding sites for

the active polyamide, and DNA alkylation within the

coding region of the gene was confirmed in cell culture

by using ligation-mediated polymerase chain reaction.

Cells treated with this polyamide-chlorambucil conjugate

did not grow in soft agar and did not form tumors

in nude mice, indicating that polyamide-treated cells

are no longer tumorigenic. The compound is active in

vivo, blocking tumor growth in mice, without any obvious

toxic effects. We extended these studies to various

cell lines representing various types of human cancers,

including solid tumors of the breast, cervix, lung, pancreas,

prostate, and bone and blood cancers, such as

leukemias. Our results suggest that polyamide-DNA

alkylators may lead to a new class of cancer chemotherapeutic

agents.

POLYAMIDES AS ACTIVATORS OF GENE EXPRESSION

In several human diseases, activation of a repressed

gene might be useful as a therapeutic approach. One

example is the neurodegenerative disease Friedreich’s

ataxia, in which gene silencing caused by an unusual

DNA structure is the primary cause of the disease. The

DNA abnormality found in 98% of patients with Friedreich’s

ataxia is the unstable hyperexpansion of a GAA

triplet repeat in the first intron of the frataxin gene,

which adopts a triplex DNA structure, resulting in

decreased transcription and reduced levels of frataxin

protein. Frataxin is a mitochondrial protein that functions

in iron homeostasis, and decreased levels of frataxin lead

to neurodegeneration and cardiomyopathies.

We designed pyrrole-imidazole polyamides to target

GAA repeats in DNA with high affinity, and we found

that these molecules relieved transcription inhibition

of the frataxin gene in cell lines and in primary lymphocytes

derived from patients with Friedreich’s ataxia.

These molecules localize in the cell nucleus, as determined

by fluorescence deconvolution microscopy with

polyamide-dye conjugates, and most likely reverse repression

of the frataxin gene by stabilizing canonical Watson-

Crick B-type DNA. Changing the sequence specificities

of the molecules abolished their ability to induce frataxin

expression. These molecules are a first step toward

therapeutic agents for treatment of Friedreich’s ataxia.

DNA RECOGNITION WITHIN CHROMATIN

Biochemical and x-ray crystallography studies indicate

that nucleosomal DNA is largely available for molec-


ular recognition by pyrrole-imidazole polyamides. Polyamide

binding sites that are located 80 bp apart on

linear DNA lie across the 2 gyres of the DNA superhelix

in the nucleosome, forming a supergroove that is

unique to the nucleosome. On the basis of this observation,

we developed bivalent pyrrole-imidazole polyamide

clamps that bind with high specificity across

the nucleosomal supergroove. X-ray crystallography

studies performed in the laboratory of our collaborator,

K. Luger, Colorado State University, indicated that the

clamps bind as designed and effectively cross-link the

2 gyres of the DNA superhelix in the nucleosome and

stabilize nucleosomal DNA from dissociation. These

molecules are useful probes of chromatin structure and

dynamics and are tools for regulation of nucleosome

mobility during transcription.

PUBLICATIONS

Beltran, A.C., Dawson, P.E., Gottesfeld, J.M. Role of DNA sequence in the binding

specificity of synthetic basic-helix-loop-helix domains. Chembiochem 6:104, 2005.

Dickinson, L.A., Burnett, R., Melander, C., Edelson, B.S., Arora, P.S., Dervan,

P.B., Gottesfeld, J.M. Arresting cancer proliferation by small-molecule gene regulation.

Chem. Biol. 11:1583, 2004.

Edayathumangalam, R.S., Weyermann, P., Dervan, P.B., Gottesfeld, J.M., Luger, K.

Nucleosomes in solution exist as a mixture of twist-defect states. J. Mol. Biol.

345:103, 2005.

Gearhart, M.D., Dickinson, L., Ehley, J., Melander, C., Dervan, P.B., Wright, P.E.,

Gottesfeld, J.M. Inhibition of DNA binding by human estrogen-related receptor 2

and estrogen receptor with minor groove binding polyamides. Biochemistry

44:4196, 2005.

Single-Molecule Conformational

Dynamics of Nucleic Acid

Enzymes

D.P. Millar, M.F. Bailey, G. Pljevalj˘cić, S. Pond, G. Stengel,

N. Tassew, E.J.C. Van der Schans

The focus of our research is the assembly and conformational

dynamics of nucleic acid–based

macromolecular machines. We use single-molecule

fluorescence methods to investigate a range of

systems, including ribozymes, DNA polymerases, and

topoisomerases. Our studies reveal the large structural

rearrangements that occur as an integral component of

the catalytic mechanism of these enzymes.

RIBOZYMES

RNA conformation plays a central role in the mechanism

of ribozyme catalysis. The hairpin ribozyme is a

small nucleolytic ribozyme that serves as a model sys-

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

tem for detailed biophysical studies of RNA folding and

catalysis. The hairpin ribozyme consists of 2 internal

loops, 1 of which contains the scissile phosphodiester

bond, displayed on 2 arms of a 4-way multihelix junction.

To attain catalytic activity, the ribozyme must fold

into a specific conformation in which the 2 loops are

docked with each other, forming a network of tertiary

hydrogen bonds. We monitor the formation of this

docked structure by using fluorescence resonance energy

transfer (FRET) and ribozyme constructs labeled with

donor and acceptor dyes. By measuring FRET at the

level of single ribozyme molecules, we reveal subpopulations

of compact and extended conformers that are

hidden in conventional experiments. Using this approach,

we found that the ribozyme populates an intermediate

state in which the 2 loops are in proximity but tertiary

interactions have yet to form. This quasi-docked state

forms rapidly (submillisecond time scale), but the subsequent

formation of tertiary contacts between the

loops occurs much more slowly. Surprisingly, the rate

of formation of tertiary structure is essentially independent

of temperature, indicating that the activation

enthalpy is negligible. Hence, the slow tertiary folding

is due to an unfavorable entropy change in reaching

the transition state.

These observations reveal that the tertiary structure

of the hairpin ribozyme is formed through a slow

conformational search process. This fundamental mechanism

of formation of RNA tertiary structure was

obscured in most previous folding studies because of

the strong propensity of RNA molecules to populate

nonnative conformations that act as kinetic traps during

the course of folding.

DNA POLYMERASES

MOLECULAR BIOLOGY 2005 187

DNA polymerases are remarkable for their ability

to synthesize DNA at rates approaching several hundred

base pairs per second while maintaining an extremely

low frequency of errors. To elucidate the origin of polymerase

fidelity, we are using single-molecule fluorescence

methods to examine the dynamic interactions

that occur between a DNA polymerase and its DNA

and nucleotide substrates. The FRET method is being

used to observe conformational transitions of the

enzyme-DNA complex that occur during selection and

incorporation of an incoming nucleotide substrate. Our

results reveal that binding of a correct nucleotide substrate

induces a slow conformational change within the

polymerase, altering the contacts between the enzyme

and the DNA primer/template. This conformational


188 MOLECULAR BIOLOGY 2005

change appears to primarily involve the finger and

thumb subdomains of the enzyme. Our studies are providing

new insights into the dynamic structural changes

responsible for nucleotide recognition and selection by

DNA polymerases. Single-pair FRET methods are also

being used to monitor the movement of the DNA primer/

template between the separate polymerizing and editing

sites of the enzyme. This active-site switching of

DNA plays a key role in the proofreading process used

to remove misincorporated nucleotides from the newly

synthesized DNA. The advantage of single-molecule

observations is that they eliminate the need to synchronize

a population of molecules, allowing these dynamic

processes to be directly observed.

TOPOISOMERASES

Topoisomerases are enzymes that control the state

of DNA supercoiling in the cell. Type I topoisomerases

introduce a nick into a strand of DNA and become

covalently joined to the cleaved strand. This process

allows the other strand to freely swivel around the first,

resulting in the relaxation of supercoils within the DNA.

The enzyme-DNA connection is then reversed, and the

broken strand is rejoined, completing the process of

supercoil removal. We are using single-pair FRET methods

to observe the DNA-unwinding activity of single

type I topoisomerase enzymes in real time. The purpose

of these studies is to directly observe DNA rotational

motions during supercoil relaxation and to

determine whether the same number of supercoils is

removed during each enzyme-DNA encounter.

PUBLICATIONS

Millar, D., Traskelis, M.A., Benkovic, S.J. On the solution structure of the T4 sliding

clamp (gp45). Biochemistry 43:12723, 2004.

Pljevalj˘cić, G., Klostermeier, D., Millar, D.P. The tertiary structure of the hairpin ribozyme

is formed through a slow conformational search. Biochemistry 44:4870, 2005.

Single-Molecule Biophysics

A.A. Deniz, S.Y. Berezhna, J.P. Clamme, A.C.M. Ferreon,

E.A. Lemke, S. Mukhopadhyay, S. Stanford, P. Zhu

We develop and use state-of-the-art singlemolecule

fluorescence methods to address

key biological questions. Single-molecule and

small-ensemble methods offer key advantages over traditional

measurements, allowing us to directly observe the

behavior of individual subpopulations in mixtures of

molecules and to measure kinetics of structural transi-

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

tions of stochastic processes under equilibrium conditions.

We use these methods to study multiple structural

states or reaction pathways and stochastic dynamics

during the folding and assembly of biomolecules.

One major goal is to apply single-molecule methods

to studies of protein and RNA folding. Using relatively

simple model systems, we are addressing several fundamental

questions about folding mechanisms. Partially

folded or misfolded protein structures are also thought

to play important cellular roles, and these states also

can be studied by using single-molecule methods. In this

context, we are examining the folding and aggregation of

synuclein, a protein implicated in the pathogenesis of

Parkinson’s disease and other neurodegenerative diseases.

We also continue to use single-pair fluorescence

resonance energy transfer (FRET) to study the folding

of RNA hairpin ribozymes, in collaboration with D.A.

Millar, Department of Molecular Biology. In addition,

we are developing a single-molecule fluorescence

quenching method that will be useful for measuring

distance changes of less than 30 Å in proteins and

RNA, a scale at which the resolution of single-pair

FRET is low.

To better study the folding, assembly, and activity of

larger and multicomponent biological complexes, we are

developing new multicolor single-molecule FRET methods.

As a first step, we developed a diffusion 3-color singlemolecule

FRET method by which 2 or more intramolecular

or intermolecular distances can be measured

simultaneously. In collaboration with J.R. Williamson,

Department of Molecular Biology, we are using these

novel methods to study the detailed mechanisms of

assembly of the bacterial ribosome. The small 30S

subunit of the ribosome assembles from a large RNA

and 21 small proteins through a complex process that

involves several steps of binding and conformational

changes. Initially, we are focusing on the conformational

properties of small RNA fragments from the 30S

subunit and on the interactions of the fragments with

their protein partners. These studies are also being

extended to the assembly of entire domains of the

30S subunit.

Finally, using a combination of high-sensitivity

imaging and fluorescence correlation spectroscopy, we

are beginning to study the lipid-mediated entry and

intracellular delivery pathways of antisense oligodeoxynucleotides

and small interfering RNA. An understanding

of these mechanisms will be critical to improving

the efficiencies of these important genetic tools.


PUBLICATIONS

Berezhna, S., Schaefer, S., Heintzmann, R., Jahnz, M., Boese, G., Deniz, A.A.,

Schwille, P. New effects in polynucleotide release from cationic lipid carriers

revealed by confocal imaging, fluorescence cross-correlation spectroscopy and single

particle tracking. Biochim. Biophys. Acta 1669:193, 2005.

Clamme, J.-P., Deniz, A.A. Three-color single-molecule fluorescence resonance

energy transfer. Chemphyschem 6:74, 2005.

Zhu, P., Clamme, J.-P., Deniz, A.A. Fluorescence quenching by TEMPO: a sub-30 Å

single molecule ruler. Biophys. J., in press.

Computer Modeling of Proteins

and Nucleic Acids

D.A. Case, M. Crowley, Q. Cui, P. Dasgupta, F. Dupradeau,*

N. Grivel,* R. Lelong,* S. Moon, D. Nguyen, D. Shivakumar,

R. Torres, R.C. Walker, L., Yan,* J. Ziegler**

* Université Jules Verne, Amiens, France

** Universität Bayreuth, Bayreuth, Germany

Computer simulations offer an exciting approach

to the study of many aspects of biochemical

interactions. We focus primarily on molecular

dynamics simulations (in which Newton’s equations of

motions are solved numerically) to model the solution

behavior of biomacromolecules. Recent applications

include detailed analyses of electrostatic interactions

in short peptides (folded and unfolded), proteins, and

oligonucleotides in solution.

In addition, molecular dynamics methods are useful

in refining solution structures of proteins by using

constraints derived from nuclear magnetic resonance

(NMR) spectroscopy, and we continue to explore new

methods in this area. Our developments are incorporated

into the Amber molecular modeling package, designed

for large-scale biomolecular simulations, and into other

software, including Nucleic Acid Builder, for developing

3-dimensional models of unusual nucleic acid structures;

SHIFTS, for analyzing chemical shifts in proteins and

nucleic acids; and RNAmotif, for finding structural motifs

in genomic sequence databases.

Additional studies on active sites of nitrogenase

and other metalloenzymes are described in the report

of L. Noodleman, Department of Molecular Biology.

NMR AND THE STRUCTURE AND DYNAMICS OF

PROTEINS AND NUCLEIC ACIDS

Our overall goal is to extract the maximum amount

of information on biomolecular structure and dynamics

from NMR experiments. To this end, we are studying

the use of direct refinement methods for determining

biomolecular structures in solution, going beyond dis-

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

tance constraints to generate closer connections between

calculated and observed spectra. We are also using

quantum chemistry to study chemical shifts and spinspin

coupling constants. Other types of data, such as

chemical shift anisotropies, direct dipolar couplings in

partially oriented samples, and analysis of cross-correlated

relaxation, are also being used to guide structure

refinement. In recent structural studies, we focused on

minor groove–binding drugs in complex with DNA and

on complexes of zinc finger proteins with RNA.

NUCLEIC ACID MODELING

MOLECULAR BIOLOGY 2005 189

Another project centers on the development of novel

computer methods to construct models of “unusual”

nucleic acids that go beyond traditional helical motifs.

We are using these methods to study circular DNA, small

RNA fragments, and 3- and 4-stranded DNA complexes,

including models for recombination sites. We continue

to develop efficient computer implementations of continuum

solvent methods to allow simplified simulations

that do not require a detailed description of the

solvent (water) molecules; this approach also provides

a useful way to study salt effects.

This research is part of a larger effort to develop

low-resolution models for nucleic acids that can be

extended to much larger structures such as circular DNA,

viruses, or models of ribosomal particles. A computer

language, NAB, was developed to make it easier to

construct and simulate molecular models for complex

and often low-resolution problems. The language is

being used to study compact and swollen viruses, to

analyze curved and circular DNA, and to simulate

assembly of ribosomes.

DYNAMICS AND ENERGETICS OF NATIVE AND

NONNATIVE STATES OF PROTEINS

Analysis methods similar to those described for

nucleic acids are also being used to estimate thermodynamic

properties of “molten globules” and unfolded

states of proteins. These studies are an extension of

our earlier work on the folding of peptide fragments of

proteins. A key feature is the development of computational

methods that can be used to model pH and

salt dependence of complex conformational transitions,

such as unfolding events.

A second aspect of this research is a detailed interpretation

of NMR results for protein nonnative states

through molecular dynamics simulations and the construction

of models for molecular motion and disorder.

In a parallel effort, we are studying correlated fluctuations

about native conformations in a variety of pro-


190 MOLECULAR BIOLOGY 2005

teins, including dihydrofolate reductase, metallo-β-lactamase,

binase, and cyclic-dependent kinase, in an

effort to make more secure connections between the

motions of proteins and the activities of enzymes.

All of these modeling activities are based on molecular

mechanics force fields, which provide estimates of

energies as a function of conformation. We continue to

work on improvements in force fields; recently, we

focused on adding aspects of electronic polarizability,

going beyond the usual fixed-charge models, and on

methods for handling arbitrary organic molecules that

might be considered potential inhibitors in drug discovery

efforts. Overall, the new models should provide

a better picture of the noncovalent interactions between

peptide groups and the groups’ surroundings, leading

ultimately to more faithful simulations.

BIOCHEMICAL SIMULATIONS AT CONSTANT pH

Like temperature and pressure, the solution pH is

an important intensive thermodynamic variable that is

commonly varied in experiments and that is used by

cells to influence biochemical function. It is now becoming

feasible to carry out practical molecular dynamics

simulations that mimic the thermodynamics of such

experiments, by allowing proton transfer between the

system of interest and a hypothetical bath of protons at

a given pH. These calculations are demanding, both

because the changes in the energetics of charge that

occur upon protonation or deprotonation must be accurately

modeled and because such simulations must

sample both molecular configurations and the large

number of protonation states that are possible in a

molecule with many acidic or basic sites.

This problem is difficult, because almost all biomolecules

have multiple sites that can bind or release

protons, and these sites are coupled to one another in

complex ways. In recent years, however, increases in

computational power and new models for estimating

the energetics of protonation and deprotonation events

have led to serious attempts at simulations that allow

the solution pH to be specified as an external variable

in a manner that parallels the ways in which temperature

or pressure are specified.

We recently developed practical methods for estimating

ionization probabilities and for allowing the

solution pH to be entered as an input variable. Figure 1

shows the results for an acidic group in the protein

thioredoxin. The curves show the distribution of energy

differences between the protonated and deprotonated

forms of the acid or base residue. We can examine the

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Fig. 1. Probability profile for the energy gap (the energy difference

between the protonated and deprotonated forms, in kcal/mol)

for the side chain of aspartic acid at position 26 in thioredoxin.

Values of λ (shown beside the curves) interpolate between the neutral

form at λ = 0 and the ionized form at λ = 1. Simple behavior

would appear as an inverted parabola; multiple conformations lead

to the more complex behavior seen at λ = 0.11.

behavior of this variable near the ionized form, corresponding

to ordinary pH, or near the neutral, protonated

form, at low pH. The results show complex

behavior at low pH, which can be analyzed and related

to the nature of the acid-base transition under those

conditions. These ideas can form the foundation of

powerful methods to explore the response of proteins

to changes in solvent pH.

PUBLICATIONS

Baker, N.A., Bashford, D., Case, D.A. Implicit solvent electrostatics in biomolecular

simulation. Adv. Macromol. Simul., in press.

Beveridge, D.L., Barreiro, G., Byun, K.S., Case, D.A., Cheatham, T.E. III, Dixit, S.B.,

Giudice, E., Lankas, F., Lavery, R., Maddocks, J.H., Osman, R., Siebert, E., Sklenar,

H., Stoll, G., Thayer, K.M., Varnai, P., Young, M.A. Molecular dynamics simulations of

the 136 unique tetranucleotide sequences of DNA oligonucleotides, 1: research

design, informatics, and results on d(CpG) steps. Biophys. J. 87:3799, 2004.

Case, D.A., Cheatham, T.E., Darden, T., Gohlke, H., Luo, R., Merz, K.M., Onufriev, A.,

Simmerling, C., Wang, B., Woods, R. The Amber biomolecular simulation programs.

J. Comput. Chem., in press.

Mongan, J., Case, D.A. Biomolecular simulations at constant pH. Curr. Opin.

Struct. Biol. 15:157, 2005.

Mongan, J., Case, D.A., McCammon, J.A. Constant pH molecular dynamics in

generalized Born implicit solvent. J. Comput. Chem. 25:2038, 2004.

Zhang., Q., Dwyer, T., Tsui, V., Case, D.A., Cho, J., Dervan, P.B., Wemmer, D.E. NMR

structure of a cyclic polyamide-DNA complex. J. Am. Chem. Soc. 126:7958, 2004.


Quantum Chemistry for

Intermediates, Reaction

Pathways, and Spectroscopy

L. Noodleman, D.A. Case, W.-G. Han, F. Himo,* T. Lovell,**

T. Liu,*** M.J. Thompson,**** R.A. Torres

* Royal Institute of Technology, Stockholm, Sweden

** AstraZeneca R&D, Mölndal, Sweden

*** University of Maryland, College Park, Maryland

**** Boston University, Boston, Massachusetts

We use a combination of modern quantum chemistry

(density functional theory) and classical

electrostatics to describe the energetics, reaction

pathways, and spectroscopic properties of enzymes

and to analyze systems with novel catalytic, photochemical,

or photophysical properties.

Critical biosynthetic and regulatory processes may

involve catalytic transformations of fairly small molecules

or groups by transition-metal centers. The ironmolybdenum

cofactor center of nitrogenase catalyzes

the multielectron reduction of molecular nitrogen to 2

ammonia molecules plus molecular hydrogen. We are

continuing our work on the catalytic cycle of this enzyme,

following up on our earlier research on the structure of

the MoFe 7 S 9 X prismane active site, where the central

ligand X most likely is nitride.

Class I ribonucleotide reductases are aerobic enzymes

that catalyze the reduction of ribonucleotides to deoxyribonucleotides,

providing the required building blocks

for DNA replication and repair. These ribonucleotideto-deoxyribonucleotide

reactions occur via a long-range

radical (or proton-coupled electron transfer) propagation

mechanism initiated by a fairly stable tyrosine

radical, “the pilot light.” When this pilot light goes

out, the tyrosine radical is regenerated by a high-oxidation-state

iron(III)-iron(IV)-oxo enzyme intermediate,

called intermediate X. We are using density functional

and electrostatics calculations in combination with

analysis of Mössbauer, electron nuclear double resonance,

and magnetic circular dichroism spectroscopic

findings to search for a proper structural and electronic

model for intermediate X. On the basis of these studies,

we propose that intermediate X contains a di-oxo

that bridges the iron(III)-iron(IV) in an asymmetric diamond

structure (Fig. 1).

In studies with E. Getzoff and M.J. Thompson,

Department of Molecular Biology, and D. Bashford, St.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 191

Fig. 1. Proposed model for the active site of class I ribonucleotide

reductase intermediate X.

Jude Children’s Hospital, Memphis, Tennessee, we are

examining the basis for the spectral tuning of the

chromophore at the active site of photoactive yellow

protein as an example of a light-activated signal transducing

protein.

In collaborations with K. Hahn, A. Toutchkine, and

D. Gremiachinsky, University of North Carolina, Chapel

Hill, North Carolina; F. Himo, Royal Institute of Technology,

Stockholm, Sweden; and M. Ullmann, University

of Bayreuth, Bayreuth, Germany, we examined the

optical properties of solvent-dependent fluorescent

dyes as prototypes for fluorescent tags that could act

as reporters of protein conformational change due to

ligand binding. These detailed calculations will be used

to improve design strategies for stable and optically

useful dyes.

Also, with Dr. Bashford’s group, we are studying

reaction pathways for the catalytic dephosphorylation

of a tyrosine side chain by a low molecular weight

protein tyrosine phosphatase. The reaction occurs in 2

distinct steps: first, formation and then hydrolysis of a

phosphocysteine intermediate.

In a collaboration with K. Janda and T. Dickerson,

Department of Chemistry, we used quantum chemical

density functional theory methods to examine the mechanism

of nornicotine-catalyzed aldol reactions in

aqueous solution. Nornicotine is a long-lived nicotine

metabolite generated under physiologic conditions in cigarette

smokers. This reaction leads to abnormal protein

glycation and to covalent modification of steroid drugs,

including the prescription corticosteroid prednisone.

We are continuing our collaboration with K.B.

Sharpless, V.V. Fokin, R. Hilgraf, and V. Rostovtsev,

Department of Chemistry, on the catalytic mechanisms

used by transition-metal ions in click chemistry, in which

metal centers catalyze ring formation from multiply


192 MOLECULAR BIOLOGY 2005

bonded precursors. Our current focus is the mechanism

of copper(I) reactions, because copper(I) in water shows

great versatility in ligating organic azides and alkynes

to form 5-membered heterocycles (triazoles) with wide

molecular diversity. On the basis of density function

theory calculations, we predict that an unusual 6-membered

copper(III) metallocycle intermediate is formed,

with only a low barrier to the triazole-copper(I) derivative,

leading to the triazole product after proteolysis.

PUBLICATIONS

Asthagiri, D., Liu, T., Noodleman, L., Van Etten, R.L., Bashford, D. On the role of the

conserved aspartate in the hydrolysis of the phosphocysteine intermediate of the low

molecular weight tyrosine phosphatase. J. Am. Chem. Soc. 126:12677, 2004.

Dickerson, T.J., Lovell, T., Meijler, M.M., Noodleman, L., Janda, K.D. Nornicotine

aqueous aldol reactions: synthetic and theoretical investigations into the origins of

catalysis. J. Org. Chem. 69:6603, 2004.

Himo, F., Lovell, T., Hilgraf, R., Rostovtsev, V.V., Noodleman, L., Sharpless, K.B.,

Fokin, V.V. Copper(I)-catalyzed synthesis of azoles: DFT study predicts unprecedented

reactivity and intermediates. J. Am. Chem. Soc. 127:210, 2005.

Theoretical and Computational

Molecular Biophysics

C.L. Brooks III, C. An, R. Armen, I. Borelli, D. Bostick,

S.R. Brozell, D. Braun, L. Bu, J. Chen, M.F. Crowley,

O. Guvench, R. Hills, W. Im, J. Khandogin, I. Khavrutskii,

J. Lee, R. Mannige, M. Michino, H.D. Nguyen, Y.Z. Ohkubo,

M. Olson,* S. Patel, D.J. Price, V. Reddy, H.A. Scheraga,**

C. Shepard, A. Stoycheva, F.M. Tama, M. Taufer,***

K.A. Taylor,**** I.F. Thorpe, C. Wildman

* U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick,

Maryland

** Cornell University, Ithaca, New York

*** University of Texas, El Paso, Texas

**** Florida State University, Tallahassee, Florida

Understanding the forces that determine the

structure of proteins, peptides, nucleic acids,

and complexes containing these molecules and

the processes by which the structures are adopted is

essential to complete our knowledge of the molecular

nature of structure and function. To address such questions,

we use statistical mechanics, molecular simulation,

statistical modeling, and quantum chemistry.

Creating atomic-level models to simulate biophysical

processes (e.g., folding of a protein or binding of a

ligand to a biological receptor) requires (1) the development

of potential energy functions that accurately

represent the atomic interactions and (2) the use of

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

quantum chemistry to aid in determining the parameters

for the models. Calculation of thermodynamic properties

requires the development and implementation of

new theoretical and computational approaches that connect

averages over atomistic descriptions to experimentally

measurable thermodynamic and kinetic properties.

Interpreting experimental results at more microscopic

levels is fueled by the development and investigation

of theoretical models of the processes of interest.

Massive computational resources are needed to realize

these objectives, and this need motivates our efforts

aimed at the efficient use of new computer architectures,

including large supercomputers, Linux Beowulf

clusters, and computational grids. Each of the objectives

and techniques mentioned represents an ongoing

area of development within our research program in

computational biophysics. The following are highlights

of a few specific projects.

FOLDING, STRUCTURE, AND FUNCTION OF

MEMBRANE-BOUND PROTEINS

Folding, insertion, and stability of membrane proteins

are directly governed by the unique hydrophilic

and hydrophobic environment provided by biological

membranes. Modeling this heterogeneous environment

is both an obstacle and an essential requisite to experimental

and computational studies of the structure and

function of membrane proteins. Because of the biological

importance and marked presence of membrane

proteins in known genomes (i.e., they account for

about 30% of all proteins), one aim of modern molecular

biophysics should be the development of methods

that can be used in experimental studies to understand

the structure and function of these systems. We recently

developed theoretical methods that enable the exploration

of protein insertion and folding in membranes.

These methods combine the sampling methods of

replica-exchange molecular dynamics with novel generalized

Born implicit solvent/implicit membrane continuum

electrostatic theories.

We recently used de novo folding–membrane association–insertion

simulations of a series of peptides

(tryptophan-flanked α-helical peptides) designed to

explore the concept of hydrophobic mismatch in modulating

folding and membrane insertion. Using the simulations,

we examined the detailed molecular mechanism

of peptide insertion into biological membranes. Our

results indicated a common mechanism for the insertion

of transmembrane helices of relatively hydrophobic

sequences. As illustrated in Figure 1, a peptide


ecomes associated with the membrane interface, transferring

from the aqueous phase, and then helical structure

begins to form. The fluctuating helical structure in

the interfacial peptide grows until a critical helical length

is achieved, and the peptide then inserts via its N-terminal

end to form a transmembrane helix. These findings

suggest an emerging potential for the de novo investigation

of integral membrane peptides and proteins and

a mechanism to assist in experimental approaches to

characterizing and determining the structure of these

important systems.

Fig. 1. Mechanism of membrane association, folding, and insertion

of a designed membrane peptide. The headgroup regions of the

membrane are schematically represented by the parallel plates; the

lipid tail–group regions, by the intervening space. Peptides first move

from an aqueous environment above the membrane to the interfacial

region, where they begin to form helical structure. When the

fluctuating helical structure reaches a critical value near 70%–80%

helix, the peptide spontaneously inserts from its N-terminal end.

LARGE-SCALE FUNCTIONAL DYNAMICS IN

MOLECULAR ASSEMBLIES

Many naturally occurring “machines,” such as

ribosomes, myosin, and viruses, require large-scale

dynamical motions as a component of their normal

functioning. These motions involve the “mechanical”

reorganization of major parts of the structure of the

machine in response to binding of effectors or to the

addition of energy in the form of thermal fluctuations

or provided by chemical catalysis. Exploring and understanding

the character and nature of such large-scale

reorganization of biological machines are ongoing goals

in our laboratory. Using theoretical approaches derived

from the treatment of mechanoelastic materials, we

are constructing theoretical models for the motions of

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 193

large molecular assemblies, including viral capsids,

ribosomes, and myosin.

In the life cycle of viruses, large-scale reorganization

of the protein-protein interfaces of the viral capsid

coat is necessary for the functioning of the virus. These

motions involve the overall swelling (or shrinking) of

the capsid as it reveals (or sequesters) its genome.

How such large conformational changes occur is key

to understanding and potentially controlling aspects of

viral infectivity. Using theoretical methods called elastic

network normal mode analysis, we explored putative

swelling and shrinking transitions for a number of

icosahedral viral capsids of various complexity, from

T-numbers of 1 to 13. We discovered a surprisingly

similar mechanism for particle expansion and shrinking,

despite the significant variation of individual capsid

architectures. We examined the collective modes

of motion that were energetically easiest to excite,

while also directing the conformational change between

a swollen (or contracted) icosahedrally symmetric conformation,

as observed experimentally.

Our calculations (Fig. 2) show that the lowest energy

modes that lead to swollen (compressed) states, despite

the complexity of the underlying capsid architecture as

indicated by the T-number, involves one key mode that

produces a uniform deformation of the entire capsid

and another that predominately distorts the structures

around the 5-fold symmetry axes. Because the mechanical

properties, and the global level of deformations

necessary for viral functioning, appear to depend solely

on the shape of the viral particle, we can hypothesize

general mechanisms for a number of viral functions,

Fig. 2. Displacement directions for the swelling of the capsid of

the bacteriophage HK97 during maturation from the prohead II state

to the head II state as calculated by using elastic network normal

mode analysis. The amplitude and direction of motion are indicated

by the arrows. The first mode (A) accounts for nearly uniform displacement

of all protein units in the capsid, whereas the next lowest

energy mode (B) promotes “bulging” around the 5-fold axes of

the capsid.


194 MOLECULAR BIOLOGY 2005

from the transfer of genetic material to a host system

to the encapsulation of this genetic material in the

assembly and maturation of viruses.

PUBLICATIONS

Chen, J., Brooks, C.L. III, Wright, P.E. Model-free analysis of protein dynamics:

assessment of accuracy and model selection protocols based on molecular dynamics

simulation. J. Biomol. NMR 29:243, 2004.

Chen, J., Im, W., Brooks, C.L. III. Refinement of NMR structures using implicit solvent

and advanced sampling techniques. J. Am. Chem. Soc. 126:16038, 2004.

Chen, J., Won, H.S., Im, W., Dyson, H.J., Brooks, C.L. III. Generation of nativelike

protein structures from limited NMR data, modern force fields and advanced

conformational sampling. J. Biomol. NMR 31:59, 2005.

Dominy, B.N., Minoux, H., Brooks, C.L. III. An electrostatic basis for the stability

of thermophilic proteins. Proteins 57:128, 2004.

Falke, S., Tama, F., Brooks, C.L. III, Gogol, E.P., Fisher, M.T. The 13 Å structure

of a chaperonin GroEL-protein substrate complex by cryo-electron microscopy. J.

Mol. Biol. 348:219, 2005.

Feig, M., Brooks, C.L. III. Recent advances in the development and application of

implicit solvent models in biomolecule simulations. Curr. Opin. Struct. Biol.

14:217, 2004.

Feig, M., Im, W., Brooks, C.L. III. Implicit solvation based on generalized Born

theory in different dielectric environments. J. Chem. Phys. 120:903, 2004.

Feig, M., Onufriev, A., Lee, M.S., Im, W., Case, D.A., Brooks, C.L. III. Performance

comparison of generalized Born and Poisson methods in the calculation of electrostatic

solvation energies for protein structures. J. Comput. Chem. 25:265, 2004.

Ferrara, P., Gohlke, H., Price, D.J., Klebe, G., Brooks, C.L. III. Assessing scoring

functions for protein-ligand interactions. J. Med. Chem. 47:3032, 2004.

Guvench, O., Brooks, C.L. III. Efficient approximate all-atom solvent accessible

surface area method parameterized for folded and denatured protein conformations.

J. Comput. Chem. 25:1005, 2004.

Guvench, O., Brooks, C.L. III. Tryptophan side chain electrostatic interactions

determine edge-to-face vs parallel-displaced tryptophan side chain geometries in

the designed β-hairpin ”trpzip2.” J. Am. Chem. Soc. 127:4668, 2005.

Guvench, O., Price, D.J., Brooks, C.L. III. Receptor rigidity and ligand mobility in

trypsin-ligand complexes. Proteins 58:407, 2005.

Im, W., Brooks, C.L. III. Interfacial folding and membrane insertion of designed

peptides studied by molecular dynamics simulations. Proc. Natl. Acad. Sci. U. S. A.

102:6771, 2005.

Karanicolas, J., Brooks, C.L. III. An evolution of minimalist models for protein folding:

from the behavior of protein-like polymers to protein function. Biosilico 2:127, 2004.

Mackerell, A.D., Jr., Feig, M., Brooks, C.L. III. Extending the treatment of backbone

energetics in protein force fields: limitations of gas-phase quantum mechanics

in reproducing protein conformational distributions in molecular dynamics simulations.

J. Comput. Chem. 25:1400, 2004.

Natrajan, A., Crowley, M., Wilkins-Diehr, N., Humphrey, M.A., Fox, A.D., Grimshaw,

A.S., Brooks, C.L. III. Studying protein folding on the Grid: experiences using CHARMM

on NPACI resources under Legion. Concurr. Comput. Pract. Exp. 16:385-397, 2004.

Patel, S., Brooks, C.L., III. A nonadditive methanol force field: bulk liquid and liquid-vapor

interfacial properties via molecular dynamics simulations using a fluctuating

charge model. J. Chem. Phys. 122:24508, 2005.

Patel, S., Mackerell, A.D., Jr., Brooks, C.L. III. CHARMM fluctuating charge force

field for proteins, 2: protein/solvent properties from molecular dynamics simulations

using a nonadditive electrostatic model. J. Comput. Chem. 25:1504, 2004.

Price, D.J., Brooks, C.L. III. A modified TIP3P water potential for simulation with

Ewald summation. J. Chem. Phys. 121:10096, 2004.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Stoycheva, A.D., Brooks, C.L. III, Onuchic, J.N. Gatekeepers in the ribosomal protein

S6: thermodynamics, kinetics, and folding pathways revealed by a minimalist

protein model. J. Mol. Biol. 340:571, 2004.

Tama, F., Brooks, C.L. III. Diversity and identity of mechanical properties of icosahedral

viral capsids studied with elastic network normal mode analysis. J. Mol.

Biol. 345:299, 2005.

Tama, F., Feig, M., Liu, J., Brooks, C.L. III, Taylor, K.A. The requirement for

mechanical coupling between head and S2 domains in smooth muscle myosin

ATPase regulation and its implications for dimeric motor function. J. Mol. Biol.

345:837, 2005.

Tama, F., Miyashita, O., Brooks, C.L. III. Normal mode based flexible fitting of

high-resolution structure into low-resolution experimental data from cryo-EM. J.

Struct. Biol. 147:315, 2004.

Taufer, M., Crowley, M., Price, D.J., Chien, A.A., Brooks, C.L. III. Study of a highly

accurate and fast protein-ligand docking method based on molecular dynamics.

Concurr. Comput. Pract. Exp., in press.

Thorpe, I.F., Brooks, C.L. III. The coupling of structural fluctuations to hydride

transfer in dihydrofolate reductase. Proteins 57:444, 2004.

Computation and Visualization

in Structural Biology

A.J. Olson, D.S. Goodsell, M.F. Sanner, A. Gillet, Y. Hu,

R. Huey, C. Li, S. Karnati, W. Lindstrom, G.M. Morris,

A. Omelchenko, M. Pique, B. Norledge, R. Rosenstein,

D. Stoffler, Y. Zhao

In the Molecular Graphics Laboratory, we develop

novel computational methods to analyze, understand,

and communicate the structure and interactions

of complex biomolecular systems. This past year,

we showed the effectiveness of 3-dimensional molecular

models as a tangible human-computer interface in

educational and research settings. Within our component-based

visualization environment, we continue to

develop methods for predicting biomolecular interactions,

analyzing biomolecular structure and function,

and presenting the biomolecular world in education

and outreach.

We have applied these methods to several important

systems in human health and welfare. We continue

the search for inhibitors of HIV protease to fight

the growing problem of drug resistance in HIV disease.

We used AutoDock, a suite of programs for predicting

bound conformations and binding energies for biomolecular

complexes, in the virtual screening of large databases

of compounds and ultimately identified new compounds

for use in the treatment of cancer. We used methods for

predicting protein interactions to probe the mechanism

of blood coagulation.


TANGIBLE INTERFACES FOR STRUCTURAL BIOLOGY

We are using the evolving technology of computer

autofabrication (“3-dimensional printing”) to produce

physical models of complex molecular assemblies (Fig. 1).

With this technology, a physical model based on a virtual

computer model is built up layer by layer. The great

advantage of autofabrication is that nearly any shape

can be built; the shape is limited only by the imagination

of the researcher and the structural integrity of the

building material. We have used 2 technologies: 1 that

is much like using a hot glue gun, in which the model is

built from layers of molten plastic, and 1 in which gypsum

powder and colored binders applied with an ink jet

technology are used to create full-color models.

Fig. 1. A sample of the molecular models built by using automated

fabrication techniques shows a wide range of molecular representations,

scales, and sizes.

In collaboration with the Human Interfaces Technology

Laboratory at the University of Washington, Seattle,

Washington, we developed an augmented reality environment

that embeds these 3-dimensional models within

the virtual environment of the computer. The goal of this

technology is to create a sense of user presence in a

computational interaction, combining the intuitive tactile

interaction of model manipulation with the rich bioinformatics

and visualization tools that are available in

the computer environment. As shown in Figure 2, the

augmented reality environment tracks the position of the

model, displaying a video image of the model and user

and overlaying a computer-generated image that is spatially

registered with the model as the user manipulates

and explores the structure. In tests of the model, high

school and college students reported that they experienced

a compelling sense of realism of the virtual object

and enhanced interaction with the subject matter.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 195

Fig. 2. Top, The augmented reality environment. The user holds

the model under a FireWire camera. Bottom, A video image of the

model is displayed on the computer screen, with an overlaid computer-generated

image. Here, the electrostatic potential and field of

superoxide dismutase are shown with volume-rendered clouds and

small animated arrows.

We use the program Python Molecule Viewer to

create a diverse range of different representations for

both our virtual molecular objects and our tangible models,

simplifying integration of the models with the virtual

environment. Python Molecule Viewer allows us to

combine backbone representation, atomic representations,

and surfaces and to incorporate markers for spatial

tracking. We are also using computer-aided design

and manufacturing methods to design mechanical connectors

and magnetic fittings that incorporate aspects

of flexibility and interaction into the models. Vision,

the visual programming interface, is used to integrate

nonmolecular features and properties, such as electrostatics

and hydrophobicity, into the virtual and physical

environment.

COMPONENT-BASED VISUALIZATION ENVIRONMENT

To facilitate the integration and interoperation of

computational models and techniques from a wide


196 MOLECULAR BIOLOGY 2005

variety of scientific disciplines, we continue to expand

our component-based software environment. The environment

is centered on Python, a high-level, object-oriented,

interpretive programming language. This approach

allows the compartmentalization and reuse of software

components. Python provides a powerful “glue” for

assembling computational components and, at the same

time, a flexible language for the interactive scripting of

new applications.

We recently added a visual programming environment,

Vision, that supports the interactive and visual

combination of computational nodes into networks that

correspond to algorithms coded at a high level (Fig. 3).

Vision provides nonprogrammers an intuitive interface

for building networks that describe new computational

pipelines and novel visualizations of data. The basic

molecular visualization methods of Python Molecule

Viewer, a molecular symmetry generator, and a volumerendering

method are a few of the currently available

nodes, and new nodes are easy to create in the Python

language. The combination of the visual programming

model and the ability to interactively inspect and edit

nodes written in a high-level language creates an unprecedented

number of levels at which users can interact

with the program. The software tools developed by

using our software components have been distributed

to more than 10,600 users, with an average of 250

downloads a month during the past year.

We released a new version of our software tools in

December 2004 that contains a large number of improve-

Fig. 3. Vision, a visual programming environment, allows users

to build networks of visualization software, creating new computational

pipelines and novel visualizations of data. The canvas is shown

at the center, where users interactively combine computational nodes.

The network shown is a visualization of an electron micrograph

reconstruction of a virus, colored by the radial depth and with a

sector removed to show the interior structure.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

ments and additions. In particular, we streamlined our

distribution mechanism and included concurrent versioning

system entries that allow users to update the

software once it has been installed. We fixed several

bugs and added new packages, including mesh decimation

algorithms and support for manipulating and

visualizing volumetric data. In addition, we increased

the number of tests that are run on a nightly basis to

more than 2500.

MODELING OF FLEXIBILITY

In a project funded by the National Institutes of

Health, we developed Flexibility Tree, a hierarchical and

multiresolution representation of the flexibility of biological

macromolecules that can be used in computational

simulations. With this software, a user can encode a

small subset of a protein’s conformational subspace.

After implementing the core infrastructure of Flexibility

Tree and integrating it with Python Molecule Viewer and

Vision, we are building such trees for molecular systems,

including HIV type 1 protease and protein kinases.

A number of laboratories around the world have

developed software tools for extracting the information

that describes how the various parts of proteins move

relative to each other. We are now using Flexibility Tree

to assess the quality of the decomposition of the protein

structure into rigid bodies provided by these tools

as well as the accuracy of the motions calculated by

using these methods. Early results indicate that when

small local perturbations are allowed in addition to the

motions predicted by these tools, the Flexibility Tree

covers a conformational space that includes both open

and closed conformations of our test systems with

accuracy sufficient for docking experiments. Our next

step will be to design prototype docking tools that can

include protein flexibility based on the Flexibility Tree.

VIRTUAL SCREENING WITH AUTODOCK

We have developed new interactive tools to streamline

the process of virtual screening in AutoDock. With

these tools, users can perform docking experiments to

evaluate the binding of a database of molecules with a

particular macromolecule of interest. In collaboration

with I.A. Wilson, Department of Molecular Biology, we

used the method to discover new inhibitors for aminoimidazole

carboxamide ribonucleotide transformylase, a

target for new cancer chemotherapeutic agents. The

diversity set from the National Cancer Institute was

screened, and 44 potential candidates were identified.

In vitro inhibition assays indicated that 8 of the 44

were soluble compounds, had chemical scaffolds that


differed from the general folate template, and caused

inhibition when used in micromolar concentrations.

Currently, we are optimizing the lead candidates; our

goal is to obtain novel nonfolate inhibitors.

AutoDock is currently used in more than 3200

academic and commercial laboratories worldwide. We

continued development of AutoDock by testing a new

empirical free-energy force field. The force field incorporates

a charge-based model for evaluation of hydrophobicity

and an improved method for evaluating the

geometry of hydrogen bonding. The force field was

calibrated by using a set of 138 protein complexes of

known structure taken from the Ligand Protein Database

from the laboratory of C.L. Brooks, Department

of Molecular Biology. We anticipate that the revised

AutoDock, which incorporates this new force field and

methods for selective flexibility in the protein target, will

be released in 2005.

We also used AutoDock to predict intermolecular

interactions in several biological systems. In collaboration

with C.F. Barbas, Department of Molecular Biology,

we investigated the binding of peptides to the catalytic

aldolase antibody 93F3. To explore the large conformational

space available to these peptides, we used a

divide-and-conquer approach that separates the search

space into searchable blocks. In studies with G. Legge,

University of Texas, Austin, Texas, we explored the

interaction between the cytoplasmic tail of tissue factor

and the WW domain of proline isomerase PIN1,

focusing on the interaction of several key phosphoserine

residues.

FIGHTING DRUG RESISTANCE IN HIV DISEASE

We are continuing our work on inhibitors to fight

drug resistance in the treatment of AIDS (Fig. 4). In

collaboration with K.B. Sharpless and C.-H. Wong,

Department of Chemistry, we have focused on the

design of inhibitors that assemble within the active

Fig. 4. The predicted bound conformation of sanguinarine, a potential

lead compound for the development of novel HIV protease

inhibitors.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 197

site of HIV protease. We showed that the triazole

formed in the click chemistry reaction is an effective

mimic for the peptide group in traditional inhibitors,

forming similar hydrogen-bonding interactions.

Currently, we are moving the FightAIDS@Home

system from an outside provider to a new server strategy

that will be implemented in the Molecular Graphics

Laboratory. FightAIDS@Home enlists the worldwide

community in a large computational effort to design

effective therapeutic agents to fight AIDS. Personal

computers are used in the program when the computers

are not in use by their owners, providing an enormous,

and largely untapped, computational resource.

The current goal is to identify inhibitors that are effective

against the wild-type virus and against common

mutant forms of the virus. The large computational

resources provided by FightAIDS@Home enables the

screening of large databases of compounds and use

of multiple mutant targets, allowing estimation of the

potential of a compound to remain effective when viral

mutations occur that cause resistance to drugs currently

used to treat HIV disease.

PREDICTING PROTEIN-PROTEIN INTERACTIONS

With the goal of creating a comprehensive tool for

predicting protein-protein interactions, we incorporated

both SurfDock and AutoDock into the Python programming

environment. SurfDock uses a variable-resolution

spherical harmonics representation to find candidate

orientations, and AutoDock is then used to explore local

atomic rearrangements at the interface. We tested the

method on a set of 59 protein-protein complexes of

known structure and optimized the level of smoothing

used in the spherical harmonics approximation of the

molecular surfaces. The results of the docking test

depended on the force field used to score possible orientations.

The best results were obtained with a residue-based

pair-wise potential of mean force.

VISUAL METHODS FROM ATOMS TO CELLS

Understanding structural molecular biology is essential

to foster progress and critical decision making among

students, policy makers, and the general public. In the

past year, we continued our longstanding commitment

to science education and outreach with a combination

of presentations, popular and professional illustrations

and animation, 3-dimensional tangible models, and a

presence on the Worldwide Web. In these projects, we

use the diverse visualization tools developed in the

Molecular Graphics Laboratory to disseminate results

that range from atomic structure to cellular function.


198 MOLECULAR BIOLOGY 2005

We created a 3-dimensional model that demonstrates

viral assembly. The model is composed of pentamers

from the structure of poliovirus, with embedded magnets

on the interacting faces. When 12 or more of

these pentamer models are placed in a closed container

and gently shaken, they self-assemble in a matter of

seconds to form a spherical capsid.

We also continued several regular features that

informally present molecular structure and function.

The “Molecule of the Month” at the Protein Data Bank

(http://www.rcsb.org/pdb) provides an accessible introduction

to this central database of biomolecular structure.

Each month, a new molecule is presented with a

description of its structure, function, and relevance to

health and welfare (Fig. 5). Visitors are then given suggestions

about how to begin their own exploration of

the structures in the data bank. Currently, we are collaborating

with T. Herman, Milwaukee School of Engineering,

Milwaukee, Wisconsin, to combine material

from the “Molecule of the Month” with 3-dimensional

models and multimedia tutorials to create educational

modules for use at high school and college levels. Other

projects include “The Molecular Perspective,” articles

in the journal The Oncologist that present structures

of interest to clinical oncologists and provide a source

of continuing education for physicians, and “Recognition

in Action,” a new series in the Journal of Molecular

Recognition.

Fig. 5. Three different types of catalase. Catalase was presented

as a Molecule of the Month in 2004 after a request from a high

school teacher.

PUBLICATIONS

Berman, H.M., Ten Eyck, L.F., Goodsell, D.S., Haste, N.M., Kornev, A. Taylor,

S.S. The cAMP binding domain: an ancient signaling module. Proc. Natl. Acad.

Sci. U. S. A. 102:45, 2005.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Brik, A., Alexandros, J., Lin, Y.-C., Elder, J.H., Olson, A.J., Wlodawer, A., Goodsell,

D.S., Wong, C.-H. 1,2,3-Triazole as a peptide surrogate in the rapid synthesis

of HIV protease inhibitors. Chembiochem 6:1167, 2005.

Gillet, A., Sanner, M., Stoffler, D., Goodsell, D.S., Olson, A.J. Augmented reality

with tangible auto-fabricated models for molecular biology applications. In: IEEE

Visualization: Proceedings of the Conference on Visualization ’04. IEEE Computer

Society, Washington, DC, 2004, p. 235.

Gillet, A., Sanner, M., Stoffler, D., Olson, A. Tangible augmented interfaces for

structural molecular biology. IEEE Comput. Graph. Appl. 25:13, 2005.

Gillet, A., Sanner, M., Stoffler, D., Olson, A. Tangible interfaces for structural

molecular biology. Structure (Camb.) 13:483, 2005.

Goodsell, D.S. Computational docking of biomolecular complexes with AutoDock. In:

Protein-Protein Interactions: A Molecular Cloning Manual, 2nd ed. Golemis, E., Adams,

P. (Eds.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, in press.

Goodsell, D.S. The molecular perspective: cyclins. Oncologist 9:592, 2004; Stem

Cells 22:1121, 2004.

Goodsell, D.S. The molecular perspective: cytochrome c and apoptosis. Oncologist

9:226, 2004; Stem Cells 22:428, 2004.

Goodsell, D.S. The molecular perspective: L-asparaginase. Oncologist 10:238,

2005; Stem Cells 23:710, 2005.

Goodsell, D.S. The molecular perspective: major histocompatibility complex.

Oncologist 10:80, 2005; Stem Cells 23:454, 2005.

Goodsell, D.S. The molecular perspective: morphine. Oncologist 9:717, 2004;

Stem Cells 23:144, 2005.

Goodsell, D.S. The molecular perspective: nicotine and nitrosamines. Oncologist

9:353, 2004; Stem Cells 22:645, 2004.

Goodsell, D.S. The molecular perspective: polycyclic aromatic hydrocarbons.

Oncologist 9:469, 2004; Stem Cells 22:873, 2004.

Goodsell, D.S. Recognition in action: flipping pyrimidine dimers. J. Mol. Recognit.

18:193, 2005.

Goodsell, D.S. Representing structural information. In: Current Protocols in Bioinformatics.

Baxeranis, A.D., Davison, D.B. (Eds.). Wiley & Sons, Hoboken, NJ, in press.

Goodsell, D.S. Visual methods from atoms to cells. Structure (Camb.) 13:347, 2005.

Li, C., Xu, L., Wolan, D.W., Wilson, I.A., Olson, A.J. Virtual screening of human

5-aminoimidazole-4-carboxamide ribonucleotide transformylase against the NCI

diversity set by use of AutoDock to identify novel nonfolate inhibitors. J. Med.

Chem. 47:6681, 2004.

Sanner, M.F. A component-based software environment for visualizing large macromolecular

assemblies. Structure (Camb.) 13:447, 2005.

Sanner, M.F. Using the Python programming language for bioinformatics. In: Encyclopedia

of Genetics, Genomics, Proteomics and Bioinformatics. Jorde, L.B., Little,

P.F.R., Dunn, M.J., et al. (Eds.). Wiley & Sons, Hoboken, NJ, in press.

Zhu, X., Tanaka, F., Hu, Y., Heine, A., Fuller, R., Zhong, G., Olson, A.J., Lerner,

R.A., Barbas, C.F. III, Wilson, I.A. The origin of enantioselectivity in aldolase antibodies:

crystal structure, site-directed mutagenesis, and computational analysis. J.

Mol. Biol. 343:1269, 2004.


Computational Structural

Proteomics and Ligand Discovery

R. Abagyan, J. An, A. Cheltsov, A. Bordner,* C. Cavasotto,*

J. Kovacs, J. Fernandez-Recio,** M. Totrov,* X. Zhang,***

M. Dawson,*** A. McCluskey,**** B. Marsden*****

* Molsoft L.L.C., La Jolla, California

** Institut de Recerca Biomèdica, Barcelona, Spain

*** Burnham Institute, La Jolla, California

**** University of Newcastle, Callaghan, Australia

***** Structural Genomics Consortium, Oxford, England

Every day about 15 new crystal structures are

deposited in the Protein Data Bank. The 30,000

molecular structures in the bank contain rich information

about protein function and provide a unique

opportunity for rational search for or design of small

molecules that can be used as therapeutic agents. We

use computational structural proteomics, bioinformatics,

molecular mechanics, and cheminformatics to

characterize the function of proteins and to design

molecular structures.

Traditionally, we have focused on accurate docking

and screening of small molecules and have used internal

coordinate mechanics to predict protein association.

In 2004, we focused on improving the information content

of evolutionary sequence conservation; predicting

and classifying ligand-binding pockets and protein-protein

interfaces; improving sequence structure alignments

for models by homology; and predicting effects of single-point

mutations, loop conformations, and protein

association geometry. We also improved protocols for

predicting receptor flexibility in ligand docking and

applied virtual screening to discover inhibitors of important

biomedical targets.

BIOINFORMATICS AND PREDICTION OF PROTEIN

FUNCTION

Functional characterization of tens of thousands of

proteins is a key computational task. To build 3-dimensional

models of structurally uncharacterized protein

sequences, we developed a procedure to accurately

align those sequences to their Protein Data Bank templates

in the areas of weak alignment. The Structural

Alignment Database of 1927 alignments was then used

to develop improved alignment/threading parameters.

Every molecular biologist is confronted with the

tasks of discovering and annotating the functions of a

protein of interest. A strong evolutionary conservation

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

measure in the context of a 3-dimensional model is a

powerful source of functional information. However,

the currently used measures have a strong dependence

on the sequence composition biases of alignments. We

developed mathematical formalism that gives a powerful

measure of sequence conservation that does not

depend on overrepresentation or underrepresentation of

certain branches in the alignment. We also used this

measure in an improved method to predict novel patches

of protein-protein interactions on protein surfaces.

Specific association of proteins is a key biological

mechanism. However, accurate prediction of interfaces

and residues involved in an interaction, often an interaction

with an unknown protein partner, is a great

challenge for most proteins or domains with known

3-dimensional structure. The preference for any particular

interface is subtle because the same surface is also

happy to be exposed to water. We attempted to solve

that problem by using more meaningful surface properties

and more sophisticated numerical methods.

Using the optimal docking area method, we showed

that with optimized desolvation parameters and an

adaptive algorithm of finding the optimal interaction

patch, the desolvation signal itself without any other

signals can be strong enough. In other studies, we

combined a desolvation signal with the improved

sequence conservation signal and used the method

successfully with a benchmark of 1496 interfaces.

PREDICTING PROTEIN STRUCTURE AND

ASSOCIATION

MOLECULAR BIOLOGY 2005 199

Predicting partial protein structure or molecular

association is a critical task in computational biology

and chemistry. This past year we proposed a method

to predict both geometry and stabilization energy for

single mutations, improved protocols for predicting protein

loops, and developed a method to predict largescale

protein movements by using simplified protein

models represented in internal coordinates.

If both partners of a protein complex are known

and their “uncomplexed” 3-dimensional models exist

or can be built, attempts can be made to predict the

association geometry (also called protein docking). In

2004, we used the internal coordinate mechanics docking

method successfully in the Critical Assessment of

Prediction of Interactions competition, partially because

of the improved docking energetics. Although in the

first round we predicted only 3 of 7 complexes, in the

second and the third rounds, we were correct in 8 of


200 MOLECULAR BIOLOGY 2005

9 tasks. We are working on further improvements of

the method.

THE CELL POCKETOME

Proteins also bind small molecules, the natural substrates

or cofactors of the proteins, or specially designed

therapeutic agents. Many orphan receptors and uncharacterized

surfaces exist. This past year, we further

optimized a pocket prediction algorithm and used it

successfully on as many as 17,000 pockets from the

Protein Data Bank. In this algorithm, a mathematical

transformation of the Lennard-Jones potential is used

to generate a potential that, contoured at a certain

level, specifically locates the potential binding sites

with a rather low level of false-positives and false-negatives

(Fig. 1).

Fig. 1. Several representatives of a predicted cell pocketome.

Using this algorithm, we predicted as many as

96.8% of experimental binding sites at an overlap level

of better than 50%. Furthermore, 95% of the predicted

sites from the apo receptors were predicted at the same

level. We showed that conformational differences between

the apo and bound pockets do not dramatically affect

the prediction results. The algorithm can be used to predict

ligand-binding pockets of uncharacterized protein

structures, suggest new allosteric pockets, evaluate the

feasibility of inhibition of protein-protein interactions,

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

and prioritize molecular targets. Finally, we collected

and classified data for the human cell pocketome, a

database of the known and the predicted binding pockets

for the human proteome structures.

The pocketome can be used for rapid evaluation of

possible binding partners of a given chemical compound.

We are using the predicted pockets to develop therapeutic

molecules that target unexpected binding pockets.

Our first result in using such a strategy was obtained in

collaboration with D.A. Lomas, University of Cambridge,

Cambridge, England; we identified the first small molecules

that block the polymerization of the Z mutant

of α 1 -antitrypsin.

COMPOUND DOCKING AND VIRTUAL LIGAND

SCREENING

Small-molecule inhibitors or activators can be discovered

rationally by carefully docking them to a target

pocket and scoring the result according to the pose

and interactions of the small molecule. The virtual

screen can be performed against millions of available

chemicals or against virtual chemically feasible molecules,

and only several dozen computationally selected

candidates need to be tested experimentally. We developed

and improved different aspects of this strategy

and applied it to different drug discovery projects. The

docking technology can also help in understanding the

structural mechanisms of the actions of small molecules

and can be used to rationally design better molecules.

Recently, we used the technology to explain the antagonistic

effect of an important class of retinoid X receptor

antagonists.

A major problem in small-molecule docking and

screening is protein flexibility and conformational

rearrangements of the binding pocket upon ligand binding.

This past year we presented several scenarios for

incorporating protein flexibility into docking calculations.

In some instances, these protocols can be used

to simultaneously predict the ligand-binding pose and

the pocket rearrangements.

PUBLICATIONS

Abagyan, R. Problems in computational structural proteomics. In: Structural Proteomics.

Sundstrom, M., Norin, M., Edwards, A. (Eds,). CRC Press, Boca Raton, FL,

in press.

An, J., Totrov, M., Abagyan, R. Comprehensive identification of “druggable” protein

ligand binding sites. Genome Inform. Ser. Workshop Genome Inform. 15:31, 2004.

An, J., Totrov, M., Abagyan, R. Pocketome via comprehensive identification and

classification of ligand binding envelopes. Mol. Cell. Proteomics 4:752, 2005.

Bordner, A.J., Abagyan, R. REVCOM: a robust Bayesian method for evolutionary

rate estimation. Bioinformatics 21:2315, 2005.


Bordner, A.J., Abagyan, R. Statistical analysis and prediction of protein-protein

interfaces. Proteins 60:353, 2005.

Bordner, A.J., Abagyan, R.A. Large-scale prediction of protein geometry and stability

changes for arbitrary single point mutations. Proteins 57:400, 2004.

Cavasotto, C.N., Kovacs, J.A., Abagyan, R.A. Representing receptor flexibility in ligand

docking through relevant normal modes. J. Am. Chem. Soc. 127:9632, 2005.

Cavasotto, C.N., Liu, G., James, S.Y., Hobbs, P.D., Peterson, V.J., Bhattacharya,

A.A., Kolluri, S.K., Zhang, X.K., Leid, M., Abagyan, R., Liddington, R.C., Dawson,

M.I. Determinants of retinoid X receptor transcriptional antagonism. J. Med.

Chem. 47:4360, 2004.

Cavasotto, C.N., Orry, A.J.W., Abagyan, R.A. The challenge of considering receptor

flexibility in ligand docking and virtual screening. Curr. Comput. Aided Drug

Des., in press.

Cavasotto, C.N., Orry, A.J.W., Abagyan, R. Receptor flexibility in ligand docking. In:

Handbook of Theoretical and Computational Nanotechnology. Reith, M., Schommers,

W. (Eds.). American Scientific Publishers, Stevenson Ranch, Calif, in press.

Fernandez-Recio, J., Abagyan, R., Totrov, M. Improving CAPRI predictions: optimized

desolvation for rigid-body docking. Proteins 60:308, 2005.

Fernandez-Recio, J., Totrov, M., Skorodumov, C., Abagyan, R. Optimal docking

area: a new method for predicting protein-protein interaction sites. Proteins

58:134, 2005.

Hill, T.A., Odell, L.R., Quan, A., Abagyan, R., Ferguson, G., Robinson, P.J.,

McCluskey, A. Long chain amines and long chain ammonium salts as novel inhibitors

of dynamin GTPase activity. Bioorg. Med. Chem. Lett. 14:3275, 2004.

Kovacs, J.A., Cavasotto, C.N., Abagyan, R.A. Conformational sampling of protein

flexibility in generalized coordinates: application to ligand docking. J. Comput.

Theor. Nanosci., in press.

Marsden, B., Abagyan, R. SAD—a normalized structural alignment database:

improving sequence-structure alignments. Bioinformatics 20:2333, 2004.

Mass Spectrometry

G. Siuzdak, J. Apon, E. Go, K. Harris, R. Lowe, A. Meyers,

A. Nordstrom, Z. Shen, C. Smith, G. Tong, S. Trauger,

W. Uritboonthai, E. Want, W. Webb, C. Wranik

METABOLITE PROFILING

Small molecules ubiquitous in biofluids are now

widely used to predict disease states. The inherent

advantage of monitoring small molecules

rather than proteins is the relative ease of quantitative

analysis with mass spectrometry. We are implementing

novel mass spectrometry and bioinformatics techniques

(Fig. 1) to investigate the metabolite profiles of

small molecules as diagnostic indicators of disease.

The ultimate goal is to develop analytical and chemical

technologies and a data management system to

identify and structurally characterize metabolites of

physiologic importance.

VIRAL CHARACTERIZATION

We have developed novel methods for characterizing

viruses that have applications to whole viruses and

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

viral proteins. Our results enabled us to examine both

local and global viral structure, gaining insight into the

dynamic changes of proteins on the viral surface.

MASS SPECTROMETRY IN SILICO

MOLECULAR BIOLOGY 2005 201

Fig. 1. A novel nonlinear approach to analyzing mass spectrometry

data for identification of metabolites.

We are also developing ultra-high-sensitivity

approaches in mass spectrometry with a new strategy

that involves pulsed laser desorption/ionization from a

silylated silicon surface. In desorption/ionization on

silicon, silicon is used to capture analytes, and laser

radiation is used to vaporize and ionize these molecules.

Using this technology, we can analyze a wide

range of molecules with unprecedented sensitivity, in

the yoctomole range (Fig. 2).

Fig. 2. Laser desorption/ionization mass spectrometry on structured

silylated silicon has sensitivity rivaling that of fluorescence.

PUBLICATIONS

Bothner, B., Taylor, D., Jun, B., Lee, K.K., Siuzdak, G., Schultz, C.P., Johnson,

J.E. Maturation of a tetravirus capsid alters the dynamic properties and creates a

metastable complex. Virology 334:17, 2005.


202 MOLECULAR BIOLOGY 2005

Go, E.P., Apon, J.V., Luo, G., Saghatelian, A., Daniels, R.H., Sahi, V., Dubrow, R.,

Cravatt, B.F., Vertes, A., Siuzdak, G. Desorption/ionization on silicon nanowires.

Anal. Chem. 77:1641, 2005.

Lacy, E.R., Wang, Y., Post, J., Nourse, A., Webb, W., Mapelli, M., Musacchio, A.,

Siuzdak, G., Kriwacki, R.W. Molecular basis for the specificity of p27 toward

cyclin-dependent kinases that regulate cell division. J. Mol. Biol. 349:764, 2005.

Lowe, R., Go, E., Tong, G., Voelcker, N.H., Siuzdak, G. Monitoring EDTA and

endogenous metabolite biomarkers from serum with mass spectrometry. Spectroscopy,

in press.

Saghatelian, A., Trauger, S.A., Want, E., Hawkins, E.G., Siuzdak, G., Cravatt,

B.F. Assignment of endogenous substrates to enzymes by global metabolite profiling.

Biochemistry 43:14332, 2004.

Want, E., Cravatt, B.F., Siuzdak, G. The expanding role of mass spectrometry in

metabolite profiling and characterization. Chembiochem, in press.

Assembly Landscape of the

30S Ribosome

J.R. Williamson, F. Agnelli, A. Beck, A. Bunner, A. Carmel,

J. Chao, S. Edgcomb, M. Hennig, E. Johnson, D. Kerkow,

E. Kompfner, K. Lehmann, H. Reynolds, W. Ridgeway,

S.P. Ryder, L.G. Scott, E. Sperling, B. Szymczyna,

M. Trevathan

The 30S ribosome is 1 of 2 subunits of the 70S

ribosome, which is responsible for the synthesis

of all proteins in bacterial cells. The 30S ribosome

is responsible for decoding the mRNA for protein synthesis.

It is composed of a large 16S RNA of approximately

1500 nucleotides and 20 small proteins (S2–S21). The

biogenesis of ribosomes consumes approximately half

of the energy of the cell in bacteria, and about 20% of

the mass of a bacterium is composed of ribosomes. Thus,

the assembly of ribosomes must be rapid and efficient.

We are using a wide variety of biophysical techniques

to study the mechanism of assembly of the 30S

ribosome in vitro. We have used nuclear magnetic resonance,

x-ray crystallography, isothermal titration calorimetry,

single-molecule fluorescence, and transient electric

birefringence to probe the details of the mechanism.

Pioneering work by Nomura led to the in vitro assembly

map for the 30S ribosome: some proteins bind independently

to the 16S rRNA, and some require prior

binding of other proteins. Using this map as a framework,

we used 30S components from Escherichia coli,

Thermus thermophilus, and Aquifex aeolicus to do

detailed studies. We have constructed an updated and

revised assembly map for the 30S subunit (Fig. 1) that

contains all of the currently available information about

the assembly pathway.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Fig. 2. The assembly landscape of the 30S subunit. The conformations

of the 16S rRNA are represented in the horizontal plane,

and the energy of the conformations is the height of the plane. Folding

of parallel pathways is indicated by the arrows. The effects of protein

binding are schematically illustrated by the 2 successive changes

in the landscape. After protein binding (circles), new downhill folding

directions are created. All parallel pathways converge on the

native 30S conformation at the bottom corner of the landscape.

The 30S unit has 3 structural domains, the 5′,

central, and 3′, and each of these has one or more

primary binding proteins that will bind independently

to RNA. This binding is followed by a wave of secondary

binding proteins for each domain and a third wave of

tertiary binding proteins. Most of the proteins have

dependencies solely within their domain; a few of the

later binding proteins have interdomain dependencies.

The assembly proceeds in a parallel manner, although

each domain has a defined hierarchy of binding order.

To probe the kinetics of the assembly of the 30S

subunit, we developed a novel assay that allows binding

of all 20 ribosomal proteins simultaneously. To

achieve this simultaneous binding, we initiate assembly

of the 30S subunit by combining 16S rRNA with a

mixture of all 20 ribosomal proteins uniformly labeled

with the stable isotope nitrogen 15. The isotopic label

does not perturb the system, but it does result in a mass

change of approximately 150 units for each protein.

After assembly proceeds for a brief period, we add an

excess of unlabeled ribosomal proteins that contain the

natural stable isotope nitrogen 14. We can readily determine

the amount of the 2 isotopes for each protein by

using mass spectrometry. By measuring this fraction

as a function of the assembly time, we can monitor the

kinetics of all proteins; we term this assay isotope pulsechase

kinetics.

Using this approach, we did an extensive analysis

of the assembly kinetics of the 30S ribosome under a

variety of conditions. We systematically varied the concentration

of the reaction, the temperature, and the

magnesium ion concentration during assembly. Using

the temperature dependence of the binding rates, we

characterized the activation energy of binding for all of

the proteins. We found that the rates of binding are

not correlated to the activation energies, and we can


monitor many different assembly steps in this complex

parallel process.

To combine all of the mechanistic information,

we have cast the assembly mechanism in terms of

an assembly landscape, which has been recently developed

in research on protein folding. The assembly

landscape of the 30S subunit (Fig. 2) shows the many

possible conformations of 16S rRNA in the horizontal

plane, and the energy of those conformations is the

height of the surface. The 30S final conformation is

located at the lower corner of the landscape, but in

the absence of ribosomal proteins, it is not the lowest

energy conformation.

Fig. 1. The revised assembly map of the 30S subunit. The 16S

ribosomal RNA is shown at the top, oriented from 5′ to 3′ direction.

Each of the arrows indicates an observed dependency of binding

for each ribosomal protein. The primary binding proteins depend

solely on interactions with 16S rRNA (top row); the secondary and

tertiary binding proteins depend on prior binding of other proteins.

The assembly proceeds in many parallel directions,

heading downhill on the landscape, and the energy of

the RNA is lowered by RNA-folding reactions that create

more RNA structure. RNA folding creates the binding

sites for the ribosomal proteins, which can then

bind, and this binding has an important consequence:

new downhill directions are created for more RNA folding.

The assembly reaction proceeds by a series of

alternating RNA conformational changes and proteinbinding

events that eventually result in the complete

assembly of the 30S subunit by the convergence of

many parallel pathways.

PUBLICATIONS

Chao, J.A., Williamson, J.R. Joint x-ray and NMR refinement of the yeast L30emRNA

complex. Structure (Camb.) 12:1165, 2004.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Klostermeier, D., Sears, P., Wong, C.-H., Millar, D.P., Williamson, J.R. A three-fluorophore

FRET assay for high-throughput screening of small-molecule inhibitors of

ribosome assembly. Nucleic Acids Res. 32:2707, 2004.

Lehmann-Blount, K.A., Williamson, J.R. Shape-specific recognition of singlestranded

RNA by the GLD-1 STAR domain. J. Mol. Biol. 346:91, 2005.

Recht, M.I., Williamson, J.R. RNA tertiary structure and cooperative assembly of a

large ribonucleoprotein complex. J. Mol. Biol. 344:395, 2004.

Ryder, S.P., Williamson, J.R. Specificity of the STAR/GSG domain protein Qk1:

implications for the regulation of myelination. RNA 10:1449, 2004.

Scott, L.G., Geierstanger, B.H., Williamson, J.R., Hennig, M. Enzymatic synthesis

and 19 F-NMR studies of 2-fluoroadenine substituted RNA. J. Am. Chem. Soc.

26:11776, 2004.

Torres, F.E., Kuhn, P., De Bruyker, D., Bell, A.G., Wolkin, M.V., Peeters, E.,

Williamson, J.R., Anderson, G.B., Schmitz, G.P., Recht, M.I., Schweizer, S.,

Scott, L.G., Ho, J.H., Elrod, S.A., Schultz, P.G., Lerner, R.A., Bruce, R.H.

Enthalpy arrays. Proc. Natl. Acad. Sci. U. S. A. 101:9517, 2004.

Nuclear Magnetic Resonance

Studies of RNA and RNA-Ligand

Complexes in Solution

M. Hennig, N. Kirchner, G.C. Pérez-Alvarado, E.P. Plant,*

J.D. Dinman*

* University of Maryland, College Park, Maryland

MOLECULAR BIOLOGY 2005 203

Viruses constantly threaten human health. Not

only are we unable to control infections caused

by old enemies such as the influenza virus, but

we are continually challenged by new enemies, such

as severe acute respiratory syndrome–associated coronavirus

(SARS-CoV). Viral mRNAs often contain signals

that tell the ribosome to change reading frames during

protein synthesis. This recoding event allows viruses

to coordinate gene expression from overlapping reading

frames such as open reading frames 1a and 1b, which

are out-of-frame coding sequences within the SARS-

CoV genome. Protein 1a is translated directly from open

reading frame 1a; the fused polyprotein 1a-1b is produced

by programmed –1 ribosomal frameshifting in

which the ribosome slips back 1 nucleotide. Like other

viral frameshift signals, the SARS-CoV signal contains

2 cis-acting mRNA elements that make up a slippery

heptanucleotide site, X XXY YYZ, followed by an adjacent

downstream 3′ pseudoknot, a stable mRNA structure.

Pseudoknots generally contain 2 stems of doublestranded

RNA and 2 or 3 loops of unpaired nucleotides.

Our biochemical and solution-state nuclear magnetic

resonance studies revealed that the pseudoknot

in the SARS-CoV frameshift signal contains 3 stems.

Mutagenesis studies indicated that specific sequences


204 MOLECULAR BIOLOGY 2005

and structures within the pseudoknot are needed for

efficient frameshifting, but the exact role of the extra

stem in the SARS-CoV frameshifting signal still remains

to be determined. Our current results suggest that the

3 stems form a complex globular RNA structure. The

elucidation of this structure via high-resolution nuclear

magnetic resonance should facilitate the rational development

of therapeutic agents designed to interfere with

SARS-CoV programmed –1 ribosomal frameshifting and

will increase our understanding of how pseudoknots

stimulate frameshifting.

We continue to develop nuclear magnetic resonance

techniques to investigate the structural and functional

diversity of RNA. Novel approaches were developed to

identify and assign 2′-hydroxyl hydrogens that exchange

rapidly with the solvent and thus are difficult to detect

in aqueous buffers. The ribose 2′-hydroxyl group distinguishes

RNA from DNA and is responsible for differences

in conformation, hydration, and thermodynamic

stability of RNA and DNA oligonucleotides. This important

group lies in the shallow groove of RNA, where it

is involved in a network of hydrogen bonds with water

molecules stabilizing RNA A-form duplexes. Structural

and dynamical information on 2′-hydroxyl protons is

essential to understand their respective roles. We provide

structural information on 2′-hydroxyl groups in the

form of orientational preferences, contradicting the

model that the 2′-hydroxyl typically points away from

the ribose H-1′ proton.

PUBLICATIONS

Hennig, M., Fohrer, J., Carlomagno, T. Assignment and NOE analysis of 2′-hydroxyl

protons in RNA: implications for stabilization of RNA A-form duplexes. J. Am. Chem.

Soc. 127:2028, 2005.

Plant, E.P., Pérez-Alvarado, G.C., Jacobs, J.L., Mukhopadhyay, B., Hennig, M.,

Dinman J.D. A three-stemmed mRNA pseudoknot in the SARS coronavirus

frameshift signal. PLoS Biol. 3:e172, 2005.

Components of the Genetic

Code in Translation, Cell

Biology, and Medicine

P. Schimmel, J. Bacher, K. Beebe, Z. Druzina, K. Ewalt,

M. Kapoor, E. Merriman, C. Motta, L. Nangle, F. Otero,

J. Reader, R. Reddy, M. Swairjo, K. Tamura, E. Tzima,

W. Waas, X.-L. Yang

The genetic code was established in the transition

from the RNA world to the theater of proteins.

The code is an algorithm, matching each

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

amino acid with a nucleotide triplet. The matching of

triplets with amino acids occurs through aminoacylation

reactions in which enzymes known as aminoacyltRNA

synthetases catalyze attachment of each amino

acid to its cognate tRNA. Each tRNA, in turn, has an

anticodon nucleotide triplet that defines the amino

acid–nucleotide triplet relationship of the code.

Each amino acid has a single tRNA synthetase.

The synthetases are thought to be among the earliest

proteins and, as such, essential components of the

translation apparatus that established the genetic code

and that were present in the last common ancestor of

the universal tree of life. As the tree developed and

branched into the 3 great kingdoms—archaebacteria,

bacteria, and eukaryotes—the enzymes were incorporated

into every cell type of every organism. During

this long evolutionary period and populating of every

cell, the enzymes adopted novel functions while keeping

their canonical role as determinates of the genetic

code. Related to their central role, the enzymes acquired

novel domains enabling them to correct errors of aminoacylation

and thereby ensure the stringent accuracy of

the code. Unrelated to their canonical activity in translation,

their expanded functions include regulation of

transcription and translation in bacteria, RNA splicing

in fungal organisms, and cytokine signaling in mammalian

cells. These novel functions connect translation

to other central pathways that control growth, development,

and regulation of all cell types.

Recently, we have focused on 2 of the expanded

functions that have connections to disease and medicine.

One function is the editing activity of the synthetases.

Mutations in the editing domain of a specific

tRNA synthetase cause ambiguity in the genetic code

and result in subtle missense substitutions in proteins

throughout the organism (Fig. 1). These changes, in

turn, cause global changes in protein function. Such

changes can, in principle, lead to specific diseases, such

as autoimmune disorders. Indeed, specific changes in

the phenotypes of mammalian cells in culture occur

when an editing-defective synthetase is present.

In mammalian cells, tyrosyl- and tryptophanyl-tRNA

synthetases are procytokines. When these synthetases

are split by alterative splicing or natural proteolysis,

specific fragments are released. These fragments are

active in signal transduction pathways. For example,

T2-TrpRS, a fragment of tryptophanyl-tRNA synthetase,

is a potent angiostatic agent. In collaborative experiments

with M. Friedlander, Department of Cell Biology,


Fig. 1. Aminoacyl-tRNA synthetases catalyze the attachment of

a noncognate amino acid onto tRNA. A distinct hydrolytic second

site prevents these substrates from being released for use in protein

synthesis. Mutations within the editing site result in the inability to

clear noncognate amino acids from the tRNA. These errors in proofreading

ultimately lead to incorporation of wrong amino acids into

a growing polypeptide. The final result of accumulation of proteins

with errors in their primary sequences is cell death.

we found that T2-TrpRS arrested angiogenesis in the

retina in neonatal mice. The fragment is so effective in

arresting angiogenesis that it is now being introduced

into a clinical setting for the treatment of blindness

caused by macular degeneration. In other research, we

are focusing on the usefulness of T2-TrpRS for treatment

of highly vascularized tumors.

To understand the antiangiogenic activity of T2-TrpRS,

we are identifying the cell signaling pathway involved.

Recent experiments indicated that vascular endothelial

cell cadherin (VE-cadherin), a calcium-dependent adhesion

molecule specifically expressed in endothelial cells

and essential for normal vascular development, binds

directly to T2-TrpRS. This binding, in turn, blocks the

proangiogenic activity of vascular endothelial cell growth

factor (Fig. 2). Currently, we are examining the mechanism

of signaling by T2-TrpRS after it is bound to VEcadherin

and the mechanism of export of T2-TrpRS from

the cytoplasm to the cell surface. In addition, on the

basis of x-ray structures, we proposed a structure-based

mechanism for cytokine activation: the structural changes

that occur when tryptophanyl- and tyrosyl-tRNA synthetases

are split into specific fragments that convert

the synthetases to cytokines.

In other research, we are investigating the critical

steps in the transition from the RNA world to the theater

of proteins. Recent findings established a plausible

scenario for the selection of L- rather than D-amino

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 205

Fig. 2. Schematic illustration of proposed model for how T2-TrpRS

(T2) interacts with VE-cadherin and blocks signaling pathways for

vascular endothelial cell growth vector (VEGF) and its receptor

(VEGFR2).

acids as the building blocks for proteins in all life forms.

Using amino acids activated in a way similar to the way

in which modern amino acids are activated, we showed

chiral-selective aminoacylation of tRNA-like molecules.

We are using x-ray analysis to understand the structural

basis of the chiral selectivity.

PUBLICATIONS

Bacher, J.M., de Crécy-Lagard, V., Schimmel, P. Inhibited cell growth and protein

functional changes from an editing-defective tRNA synthetase. Proc. Natl. Acad.

Sci. U. S. A. 102:1697, 2005.

Ewalt, K.L., Schimmel, P. Protein biosynthesis: tRNA synthetases. In: Encyclopedia

of Biological Chemistry. Lennarz, W.J., Lane, M.D. (Eds.). Academic Press, San

Diego, 2004, p. 263.

Ewalt, K.L., Yang, X.-L., Otero, F.J., Liu, J., Slike, B., Schimmel, P. Variant of

human enzyme sequesters reactive intermediate. Biochemistry 44:4216, 2005.

Metzgar, D., Bacher, J.M., Pezo, V., Reader, J., Doring, V., Schimmel, P., Marlière,

P., de Crécy-Lagard, V. Acinetobacter sp ADP1: an ideal model organism for

genetic analysis and genome engineering. Nucleic Acid Res. 32:5780, 2004.

Nordin, B.E., Schimmel, P. Isoleucyl-tRNA synthetases. In: Aminoacyl-tRNA Synthetases.

Ibba, M., Francklyn, C., Cusack, S. (Eds.). Landes Bioscience/Eurekah.com,

Georgetown, TX, 2005, p. 24.

Ribas de Pouplana, L., Musier-Forsyth, K., Schimmel, P. Alanyl-tRNA synthetases.

In: Aminoacyl-tRNA Synthetases. Ibba, M., Francklyn, C., Cusack, S. (Eds.). Landes

Bioscience/Eurekah.com, Georgetown, TX, 2005, p. 241.

Ribas de Pouplana, L., Schimmel, P. Aminoacylations of tRNAs: record-keepers for

the genetic code. In: Protein Synthesis and Ribosome Structure: Translating the

Genome. Nierhaus, K.H., Wilson, D.N. (Eds.), Wiley-VCH, New York, 2004, p. 169.

Schimmel, P. Genetic code. In: McGraw-Hill Encyclopedia of Science and Technology,

10th ed. McGraw-Hill, New York, in press.

Schimmel, P., Beebe, K. From the RNA world to the theater of proteins. In: The

RNA World, 3rd ed. Gesteland, R.R., Cech, T.R., Atkins, J.F. (Eds.), Cold Spring

Harbor Laboratory Press, Cold Spring Harbor, NY, in press.

Schimmel, P., Ewalt, K. Translation silenced by fused pair of tRNA synthetases.

Cell 119:147, 2004.


206 MOLECULAR BIOLOGY 2005

Schimmel, P., Söll, D. The world of aminoacyl-tRNA synthetases. In: AminoacyltRNA

Synthetases. Ibba, M., Francklyn, C., Cusack, S. (Eds.). Landes

Bioscience/Eurekah.com, Georgetown, TX, 2005, p. 1.

Swairjo, M.A., Schimmel, P. Breaking sieve for steric exclusion of a noncognate

amino acid from active site of a tRNA synthetase. Proc. Natl. Acad. Sci. U. S. A.

102:988, 2005.

Tamura, K., Schimmel, P. Non-enzymatic aminoacylation of an RNA minihelix with

an aminoacyl phosphate oligonucleotide. Nucleic Acids Symp. Ser. 48:269, 2004.

Tang, H.-L., Yeh, L.-S., Chen, N.-K., Ripmaster, T.L., Schimmel, P., Wang, C.-C.

Translation of a yeast mitochondrial tRNA synthetase initiated at redundant non-

AUG codons. J. Biol. Chem. 279:49656, 2004.

Tzima, E., Reader, J.S., Irani-Tehrani, M., Ewalt, K.L., Schwartz, M.A., Schimmel,

P. VE-cadherin links tRNA synthetase cytokine to anti-angiogenic function. J.

Biol. Chem. 280:2405, 2005.

Mechanisms of RNA Assembly

and Catalysis

M.J. Fedor, E.M. Calderon, J.W. Cottrell, C.P. Da Costa,

J.W. Harger, Y.I. Kuzmin, E.M. Mahen

Recent evidence that RNA catalysis participates

in regulation of gene expression as well as in

RNA processing and protein synthesis underscores

the importance of learning the molecular basis of

ribozyme activity. The hairpin ribozyme is an especially

good model for investigating RNA catalytic mechanisms

because of its relative simplicity and the availability of

high-resolution structures that provide a framework for

evaluating structure-function relationships. This ribozyme

catalyzes reversible phosphodiester cleavage

through attack of a ribose 2′ oxygen nucleophile on an

adjacent phosphorus (Fig. 1). Our goals have been to

identify which parts of the ribozyme contribute to catalysis

and to understand the chemical basis of this activity.

Like all enzymes, hairpin ribozymes combine several

strategies to enhance catalytic rate. One important

Fig. 1. Chemical mechanism of RNA cleavage mediated by the

family of small catalytic RNAs that includes the hairpin ribozyme.

Cleavage proceeds through an SN2-type mechanism that involves

in-line attack of the 2′ oxygen nucleophile on the adjacent phosphorus

to form a trigonal bipyramidal transition state in which 5

electronegative oxygen atoms form transient bonds with phosphorus.

Breaking of the 5′ oxygen-phosphorus bond generates products

with 5′ hydroxyl and 2′,3′-cyclic phosphate termini.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

strategy, which is apparent from crystal structures, is

the alignment of nucleophilic and leaving-group oxygens

in the optimal orientation for an SN 2 -type nucleophilic

attack. Biochemical and structural studies also

implicate 2 active-site nucleobases, guanine 8 and

adenine 38, in catalytic chemistry; the N-1 ring nitrogen

of guanine 8 is located near the 2′ oxygen that

acts as the nucleophile during cleavage, and the N-1

ring nitrogen of adenine 38 is located near the 5′ oxygen

leaving group.

Ribonuclease A is a protein enzyme that catalyzes

the same chemical reaction as hairpin ribozyme cleavage

and has 2 active-site histidines that occupy positions

similar to those of guanine 8 and adenine 38.

Ribonuclease A provides a textbook example of concerted

general acid-base catalysis, and the similarity

between hairpin ribozyme and ribonuclease A activesite

structures led to the idea that guanine 8 and adenine

38 might serve as general acid and base catalysts

as the histidines of ribonuclease A do. The activity of

the hairpin ribozyme increases with increasing pH,

consistent with the notion that activity depends on the

availability of guanine 8, in its unprotonated form, to

accept a proton to activate the 2′ hydroxyl nucleophile

as proposed in the general acid-base catalysis model.

However, a ribozyme variant in which guanine 8 is

replaced by an abasic residue has the same pH dependence

as an unmodified ribozyme, suggesting that the

pH transition in activity does not involve guanine 8.

These data support an alternative model in which the

protonated form of guanine 8 donates hydrogen bonds

that provide electrostatic stabilization as negative charge

develops in the transition state (Fig. 2). Replacing

adenine 38 with an abasic residue, on the other hand,

does eliminate this pH-dependent transition, evidence

that the protonation state of adenine 38 is important

for activity.

The activity that is lost when adenine 38 or guanine

8 is replaced by abasic residues can be rescued

by certain nucleobases provided in solution. The molecules

that can rescue activity all have planar structures

and an amidine group, that is, an amino group in α-position

to a ring nitrogen. The same feature is shared with

the Watson-Crick face of the missing adenine and guanine,

suggesting that chemical rescue occurs through

binding of exogenous nucleobases in the cavity left by

an abasic substitution. Purines that lack an amidine

group can inhibit chemical rescue, presumably by competing

with rescuing nucleobases for binding in the cav-


Fig. 2. Results of mechanistic studies of the hairpin ribozyme

are consistent with 2 models in which the functional form of adenine

38 is either protonated or unprotonated. In the first model

(A), protonated adenine 38 would act as a general acid by donating

a proton to the 5′ oxygen, acting in concert with hydroxide ion

that activates the 2′ oxygen nucleophile during cleavage, and unprotonated

adenine 38 would act as a general base to activate the 5′

oxygen nucleophile during ligation. In the second model (B), unprotonated

adenine 38 accepts a hydrogen bond from the 5′ hydroxyl

nucleophile during ligation and accepts a hydrogen bond from a

protonated bridging 5′ oxygen during cleavage, providing electrostatic

stabilization to developing negative charge. In both models, the

amidine group of guanine 8, in its protonated form, donates hydrogen

bonds to the 2′ and phosphoryl oxygens that stabilize the negative

charge that develops in the transition state and that position

reactive groups in the orientation appropriate for an SN2 in-line

nucleophilic attack.

ity left by the abasic substitution. Thus, rescue does not

occur through binding alone, and amidine functional

groups must form specific stabilizing interactions with

the transition state. The pH dependence of chemical

rescue of ribozymes lacking adenine 38 changes according

to the intrinsic basicity of the rescuing nucleobase.

These and other results are consistent with 2 models

of the hairpin ribozyme catalytic mechanism in which

adenine 38 contributes either general acid-base catalysis

(Fig. 2A) or electrostatic stabilization of negative charge

that develops as 5 electronegative oxygen atoms form

transient bonds with phosphorus in the transition state

(Fig. 2B).

PUBLICATIONS

Fedor, M.J., Williamson, J.R. The catalytic diversity of RNAs. Nat. Rev. Mol. Cell

Biol. 6:399, 2005.

Kuzmin, Y.I, Da Costa, C.P., Cottrell, J.W., Fedor, M.J. Role of an active site adenine

in hairpin ribozyme catalysis. J. Mol. Biol. 349:989, 2005.

Mahen, E.M., Harger, J.W., Calderon, E.M., Fedor, M.J. Kinetics and thermodynamics

make different contributions to RNA folding in vitro and in yeast. Mol. Cell

19:27, 2005.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 207

Directed Evolution of Nucleic

Acid Enzymes

G.F. Joyce, T.A. Jackson, G.C. Johns, H.R. Kalhor, C.-Y. Lai,

M. Oberhuber, B.M. Paegel, G.G. Springsteen, S.B. Voytek

All life known to exist on Earth today is based on

DNA genomes and protein enzymes, but strong

evidence indicates that it was preceded by a

simpler form of life based on RNA. This earlier era is

referred to as the “RNA world.” During that time, genetic

information resided in the sequence of RNA molecules,

and phenotype was derived from the catalytic behavior

of RNA. By studying the properties of RNA in the laboratory,

especially with regard to the evolution of catalytic

function, we can gain insight into the RNA world.

In addition, we can develop novel nucleic acid enzymes

that have applications in biology and medicine.

SYNTHESIS AND DERIVATIZATION OF RIBOSE

Ribose, the sugar component of RNA, is a minor

component among the many products of the condensation

of formaldehyde. In addition, ribose is more

reactive than most other sugars and degrades more

rapidly than they do. Thus, it is difficult to understand

why ribose is included in the genetic material.

We exploited the greater reactivity of ribose by allowing

it to react preferentially with cyanamide to form a

stable product. This product crystallized spontaneously

in aqueous solution under a broad range of conditions;

the corresponding cyanamides derived from other sugars

did not. Furthermore, the ribose-cyanamide crystals

reacted with cyanoacetylene to form cytosine α-nucleoside

in nearly quantitative yield.

The RNA-catalyzed synthesis of ribose from simple

starting materials would have been an essential reaction

in the RNA world. We approached this problem

by examining the ability of a nucleic acid template to

direct the synthesis of ribose from 2 aldehyde-bearing

oligonucleotides, one with glyceraldehyde at its 3′ end

and the other with glycoaldehyde at its 5′ end. The 2

oligonucleotides were allowed to bind at adjacent positions

along a complementary template, resulting in an

aldol reaction that gave rise to pentose sugars (Fig. 1).

No reaction was detected in the absence of the template.

Adding lysine to the mixture increased the reaction

rate substantially. This reaction will be used as

the basis for in vitro evolution experiments to obtain

RNAs that catalyze the formation of ribose.


208 MOLECULAR BIOLOGY 2005

Fig. 1. RNA-directed synthesis of pentose sugars via aldol condensation.

Two oligonucleotides, one with glyceraldehyde at its 3′

end (S1) and the other with glycoaldehyde at its 5′ end (S2), are

joined in the presence of a complementary template to form a pentose-linked

product.

CROSS-REPLICATING RNA ENZYMES

The central process of the RNA world was the RNAcatalyzed

replication of RNA. We previously developed

an RNA enzyme, termed the R3C ligase, that catalyzes

the template-directed joining of 2 RNA molecules. This

enzyme was converted to a format that allows it to

produce additional copies of itself through the joining

of 2 component subunits. The copies in turn give rise

to additional copies, resulting in an exponential increase

in the number of enzyme molecules over time. We further

modified the reaction system so that it would operate

cross-catalytically, whereby 2 RNA enzymes catalyze

each other’s synthesis from a total of 4 substrates

(Fig. 2). The newly formed copies of each enzyme give

rise to additional copies of the cross-catalytic products,

and the rate of formation of both enzymes increases

during the course of the reaction. Currently, the crossreplicating

system operates with a highly restricted set

of RNA sequences, but it provides an opportunity for

developing more efficient and more complex networks

of replicating RNAs.

CONTINUOUS EVOLUTION OF RNA ENZYMES

Previously, we developed a powerful method for the

in vitro evolution of RNA enzymes that catalyze the joining

of RNA molecules. Rather than manipulating the

RNAs through successive steps of reaction, selection,

and amplification, we devised a way to have these steps

occur continuously within a common reaction vessel.

Evolution can be carried out indefinitely by a serial transfer

procedure, whereby a small part of a completed

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Fig. 2. Cross-catalytic replication of RNA enzymes. The enzyme E

binds the substrates S1′ and S2′ and catalyzes their joining to form

the enzyme E′. Similarly, the enzyme E′ binds and joins the substrates

S1 and S2 to form the enzyme E.

reaction mixture is transferred to a new reaction vessel

that contains a fresh supply of substrates and the other

components necessary for selective amplification.

During the past year, we began 3 new lines of investigation

involving continuous in vitro evolution. First, we

modified the system so that an increased frequency of

random mutations would occur during amplification. This

modification allows us to generate and exploit genetic

diversity within the system, providing a more realistic

model of biological evolution. Second, using either 2

distinct variants of 1 enzyme or 2 different enzymes,

we sought to evolve 2 different RNA enzymes within a

common environment. These evolved enzymes will be

used to study competition and cooperation in the context

of RNA-based evolution.

Third, we implemented a novel microfluidic system

for continuous in vitro evolution. In this system, the

population of enzymes is confined to a microfluidic

circuit within a fabricated glass wafer that contains a

middle layer of an elastomeric material that functions

as control valves. The concentration of RNA is monitored

by using a confocal fluorescence microscope, and

serial transfer is triggered automatically whenever the

population size reaches a predetermined threshold. The


microfluidic system makes it possible to conduct thousands

of generations of in vitro evolution in a highly precise

manner with little intervention by the experimenter.

PUBLICATIONS

Johns, G.C., Joyce, G.F. The promise and peril of continuous in vitro evolution. J.

Mol. Evol. 61:253, 2005.

Joyce, G.F., Orgel, L.E. Progress toward understanding the origin of the RNA

world. In: The RNA World, 3rd ed. Gesteland, R.F., Cech, T.R., Atkins, J.F. (Eds.).

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, in press.

Kim, D.-E., Joyce, G.F. Cross-catalytic replication of an RNA ligase ribozyme.

Chem. Biol. 11:1505, 2004.

Paul, N., Joyce, G.F. Minimal self-replicating systems. Curr. Opin. Chem. Biol.

8:634, 2004.

Springsteen, G., Joyce, G.F. Selective derivatization and sequestration of ribose

from a prebiotic mix. J. Am. Chem. Soc. 126:9578, 2004.

Studies at the Interface of

Molecular Biology, Chemistry,

and Medicine

C.F. Barbas III, B.A. Gonzalez, L. Asawapornmongkul,

D.B. Ramachary, S. Eberhardy, R. Fuller, R. Gordley, J. Guo,

B. Henriksen, C. Lund, J. Mandell, S. Mitsumori, R. Mobini,

N.S. Chowdari, M. Popkov, D. Steiner, J. Suri, F. Tanaka,

U. Tschulena, Y. Ye, Y. Yuan, G. Zhong

We are concerned with problems in molecular

biology, chemistry, and medicine. Many of

our studies involve learning or improving on

Nature’s strategies to prepare novel molecules that

perform specific functional tasks, such as regulating a

gene, destroying cancer, or catalyzing a reaction with

enzymelike efficiency. We hope to apply these novel

insights, technologies, methods, and products to provide

solutions to human diseases, including cancer,

HIV disease, and genetic diseases.

DIRECTING THE EVOLUTION OF CATALYTIC FUNCTION

Using our concept of reactive immunization, we

have developed antibodies that catalyze aldol as well

as retro-aldol reactions of a wide variety of molecules.

The catalytic proficiency of the best of these antibodies

is almost 10 14 , a value 1000 times that of the

best catalytic antibodies reported to date and overall

the best of any synthetic protein catalyst. We have

shown the efficient asymmetric synthesis and resolution

of a variety of molecules, including tertiary and

fluorinated aldols, and have used these chiral synthons

to synthesize natural products (Fig. 1). The results

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 209

Fig. 1. A variety of compounds synthesized with the world’s first

commercially available catalytic antibody, 38C2, produced at

Scripps Research.

highlight the potential synthetic usefulness of catalytic

antibodies as artificial enzymes in addressing problems

in organic chemistry that are not solved by using natural

enzymes or more traditional synthetic methods.

To further evolve these catalytic antibodies, we are

developing genetic selection methods. Other advances

in this area include the development of the first peptide

aldolase enzymes. Using both design and selection, we

created small peptide catalysts that recapitulate many

of the kinetic features of large protein catalysts. With

these smaller enzymes, we can address how the size

of natural proteins is related to catalytic efficiency.

ORGANOCATALYSIS: A BIOORGANIC APPROACH TO

CATALYTIC ASYMMETRIC CARBON-CARBON

BOND–FORMING REACTIONS

To further explore the principles of catalysis, we

are studying amine catalysis as a function of catalytic

scaffold. Using insights garnered from our studies of

aldolase antibodies, we determined the efficacy of simple

chiral amines and amino acids for catalysis of aldol

and related imine and enamine chemistries such as

Michael, Mannich, Knoevenagel, and Diels-Alder reactions

(Fig. 2). Although aldolase antibodies are superior

in terms of the kinetic parameters, these more

simple catalysts are enabling us to quantify the importance

of pocket sequestration in catalysis.

Furthermore, many of these catalysts are cheap,

environmentally friendly, and practical for large-scale

synthesis. With this approach, we showed the scope


210 MOLECULAR BIOLOGY 2005

Fig. 2. L-Proline and other organocatalysts developed for a variety

of catalytic asymmetric syntheses via aldol, Michael, Mannich,

Diels-Alder, and Knoevenagel reactions provide access to important

classes of compounds. These catalysts make reactions that were

once complex multistep reactions, simple 1-step reactions. A wide

variety of medicinally important products can be assembled by

using the Mannich reaction manifold alone.

and usefulness of the first efficient amine catalysts of

direct asymmetric aldol, Mannich, Diels-Alder, and

Michael reactions. The organocatalyst approach is a

direct outcome of our studies of catalytic antibodies

and provides an effective alternative to organometallic

reactions that use severe reaction conditions and oftentoxic

catalysts.

We think that our discovery that the simple naturally

occurring amino acids such as L-proline and other

amines can effectively catalyze a variety of enantioselective

intermolecular reactions will change the way

many reactions will be performed. Furthermore, these

catalysts are functional in related ketone addition reactions

such as Mannich- and Michael-type reactions. As

a testament to the mild nature of this approach, we

developed the first catalytic asymmetric aldol, Mannich,

Michael, and fluorination reactions involving aldehydes

as nucleophiles. Previously, such reactions were considered

out of the reach of traditional synthetic methods.

In an extension of these concepts, we invented a

variety of novel multicomponent or asymmetric assembly

reactions (Fig. 3). Our finding that a variety of optically

active amino acids can be synthesized with proline

catalysis in which an L-amino acid begets other L-amino

acids suggests that this route may have been used in

prebiotic syntheses of optically active amino acids. In

addition, we showed that our strategy can be used to

synthesize carbohydrates directly, thereby providing a

provocative prebiotic route to the sugars essential for life.

Unlike most catalysts obtained via traditional

approaches, our catalysts are environmentally safe and

are available in both enantiomeric forms. The reactions

do not require inert conditions or heavy metals and

can be performed at room temperature without preactivation

of the donor substrates. Because amines can

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Fig. 3. A few recently developed catalytic asymmetric assembly

reactions. In these reactions, designed small organic molecules are

used to synthesize complex molecules.

act as catalysts via both nucleophilic (enamine based)

and electrophilic (iminium based) activation, they have

great potential in catalytic asymmetric synthesis.

THERAPEUTIC ANTIBODIES, IN AND OUT OF CELLS

We developed the first human antibody phage display

libraries and the first synthetic antibodies and

methods for the in vitro evolution of antibody affinity.

The ability to manipulate large libraries of human antibodies

and to evolve such antibodies in the laboratory

provides tremendous opportunities to develop new

medicines. Laboratories and pharmaceutical companies

around the world now apply the phage display

technology that we developed for antibody Fab fragments.

In our laboratory, we are targeting cancer and

HIV disease. One of our antibodies, IgG1-b12, protects

animals against primary challenge with HIV type 1

(HIV-1) and has been further studied by many other

researchers. We improved this antibody by developing

in vitro evolution strategies that enhanced its neutralization

activity. By coupling laboratory-evolved antibodies

with potent toxins, we showed that immunotoxins

can effectively kill infected cells.

We are also developing genetic methods to halt

HIV by using gene therapy. We created unique human


antibodies that can be expressed inside human cells

to make the cells resistant to HIV infection. In the

future, these antibodies might be delivered to the stem

cells of patients infected with HIV-1, allowing the development

of a disease-free immune system that would

preclude the intense regimen of antiviral drugs now

required to treat HIV disease.

Using our increased understanding of antibody-antigen

interactions, we extended our efforts in cancer therapy

and developed rapid methods for creating human

antibodies from antibodies derived from other species.

We produced human antibodies that should enable us

to selectively starve a variety of cancers by inhibiting

angiogenesis and antibodies that will be used to deliver

radionuclides to colon cancers to destroy the tumors.

We hope that some of these antibodies will be used in

clinical trials done by our collaborators at the Sloan-

Kettering Cancer Center in New York City.

On the basis of our studies on HIV-1, we used intracellular

expression of antibodies directed against angiogenic

receptors to create a new gene-based approach

to cancer. We are determining if this new approach can

be applied in vivo to halt tumor growth. Our preliminary

results indicate that this type of gene therapy can

be successfully applied to the treatment of cancer.

THERAPEUTIC APPLICATIONS OF CATALYTIC

ANTIBODIES

The development of highly efficient catalytic antibodies

opens the door to many practical applications.

One of the most fascinating is the use of such antibodies

in human therapy. We think that use of this strategy

can improve chemotherapeutic approaches to diseases

such as cancer and AIDS. Chemotherapeutic regimens

are typically limited by nonspecific toxic effects. To

address this problem, we developed a novel and broadly

applicable drug-masking chemistry that operates in

conjunction with our unique broad-scope catalytic antibodies.

This masking chemistry is applicable to a wide

range of drugs because it is compatible with virtually

any heteroatom. We showed that generic drug-masking

groups can be selectively removed by sequential

retro-aldol–retro-Michael reactions catalyzed by antibody

38C2 (Fig. 4). This reaction cascade is not catalyzed

by any known natural enzyme.

Application of this masking chemistry to the anticancer

drugs doxorubicin, camptothecin, and etoposide

produced prodrugs with substantially reduced toxicity.

These prodrugs are selectively unmasked by the

catalytic antibody when the antibody is applied at thera-

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

peutically relevant concentrations. The efficacy of this

approach has been shown in in vivo models of cancer.

Currently, we are developing more potent drugs and novel

antibodies that will allow us to target breast, colon, and

prostate cancer as well as cells infected with HIV-1. On

the basis of our preliminary findings, we think that our

approach can become a key tool in selective chemotherapeutic

strategies. To see a movie illustrating this approach,

visit http://www.scripps.edu/mb/barbas/index.html.

ADAPTOR IMMUNOTHERAPY: THE ADVENT OF

CHEMOBODIES

We think that combining the chemical diversity of

small synthetic molecules with the immunologic characteristics

of antibody molecules will lead to therapeutic

agents with superior properties. Therefore, we developed

a conceptually new device that equips small synthetic

molecules with both the immunologic effector functions

and the long serum half-life of a generic antibody molecule.

For a prototype, we developed a targeting device

based on the formation of a covalent bond of defined

stoichiometry between (1) a 1,3-diketone derivative of

an arginine–glycine–aspartic acid peptidomimetic that

targets the integrins α v β 3 and α v β 5 and (2) the reactive

lysine of aldolase antibody 38C2. The resulting

complex spontaneously assembled in vitro and in vivo,

selectively retargeted antibody 38C2 to the surface of

cells expressing integrins α v β 3 and α v β 5 , dramatically

increased the circulatory half-life of the peptidomimetic,

and effectively reduced tumor growth in animal models

of human Kaposi sarcoma and colon cancer (Fig. 5).

These studies have been extended to melanoma.

ZINC FINGER GENE SWITCHES

MOLECULAR BIOLOGY 2005 211

Fig. 4. Targeting cancer and HIV with prodrugs activated by catalytic

antibodies. A bifunctional antibody is shown targeting a cancer

cell for destruction. A nontoxic analog of doxorubicin, prodoxorubicin,

is being activated by an aldolase antibody to the toxic form

of the drug.

The solution to many diseases might be simply turning

genes on or off in a selective way. In order to pro-


212 MOLECULAR BIOLOGY 2005

Fig. 5. Adaptor Immunotherapy dramatically slows tumor growth.

A variety of cancer xenografts have been effectively treated with

chemobodies, a combination of small-molecule drugs and antibodies.

Chemobodies have characteristics that can be superior to those

of either the small molecule or the antibody alone.

duce switches that can turn genes on or off, we are

studying molecular recognition of DNA by zinc finger

proteins and methods of creating novel zinc finger DNAbinding

proteins (Fig. 6). Because of their modularity

and well-defined structural features, zinc finger proteins

are particularly well suited for use as DNA-binding proteins.

Each finger forms an independently folded domain

that typically recognizes 3 nucleotides of DNA. We

showed that proteins can be selected or designed that

contain zinc fingers that recognize novel DNA sequences.

Fig. 6. A designed polydactyl zinc finger binds 18 bp of DNA. A

single zinc finger domain is highlighted. With this direct approach,

we can construct more than a billion gene switches and use the

switches to specifically turn genes on or off in multiple organisms.

With further elaboration of the approach, every gene in the genome

can be either upregulated or downregulated, providing a new approach

to probe gene function across the genome.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

These studies are aiding the elucidation of rules for

sequence-specific recognition within this family of proteins.

We selected and designed specific zinc finger

domains that will constitute an alphabet of 64 domains

that will allow any DNA sequence to be bound selectively.

The prospects for this “second genetic code” are

fascinating and should have a major impact on basic

and applied biology.

We showed the potential of this approach in multiple

mammalian and plant cell lines and in whole

organisms. With the use of characterized modular zinc

finger domains, polydactyl proteins capable of recognizing

an 18-nucleotide site can be rapidly constructed.

Our results suggest that zinc finger proteins might be

useful as genetic regulators for a variety of human ailments

and provide the basis for a new strategy in gene

therapy. Our goal is to develop this class of therapeutic

proteins to inhibit or enhance the synthesis of proteins,

providing a direct strategy for fighting diseases

of either somatic or viral origin.

We are also developing proteins that will inhibit

the growth of tumors and others that will inhibit the

expression of a protein known as CCR5, which is a

key to infection of human cells by HIV-1. We developed

an HIV-1–targeting transcription factor that strongly

suppresses HIV-1 replication. Genetic diseases such

as sickle cell anemia are also being targeted. Using a

library of transcription factors, we developed a strategy

that effectively allows us to turn on and turn off every

gene in the genome. With this powerful new strategy,

we can quickly regulate a target gene or discover other

genes that have a key role in disease. In the future,

we hope to use novel DNA-modifying enzymes directed

by zinc fingers to manipulate chromosomes themselves.

PUBLICATIONS

Amir, R.J., Popkov, M., Lerner, R.A., Barbas, C.F. III, Shabat, D. Prodrug activation

gated by a molecular “OR” logic trigger. Angew. Chem. Int. Ed. 44:4378, 2005.

Betancort, J.M., Sakthivel, K., Thayumanavan, R., Tanaka, F., Barbas, C.F. III. Catalytic

direct asymmetric Michael reactions: addition of unmodified ketone and aldehyde

donors to alkylidene malonates and nitro olefins. Synthesis 1509, 2004, Issue 9.

Blancafort, P., Segal, D.J., Barbas, C.F. III. Designing transcription factor architectures

for drug discovery. Mol. Pharmacol. Rev. 66:1361, 2004.

Chen, E.I., Florens, L., Axelrod, F.T., Monosov, E., Barbas, C.F. III, Yates, J.R. III,

Felding-Habermann, B., Smith, J.W. Maspin alters the carcinoma proteome.

FASEB J. 19:1123, 2005.

Chowdari, N.S., Barbas, C.F. III. Total synthesis of LFA-1 antagonist BIRT-377 via

organocatalytic asymmetric construction of a quaternary stereocenter. Org. Lett.

7:867, 2005.

Chowdari, N.S., Suri, J.T., Barbas, C.F. III. Asymmetric synthesis of quaternary

α- and β-amino acids and β-lactams via proline catalyzed Mannich reactions with

branched aldehyde donors. Org. Lett. 6:2507, 2004.


Crotty, J.W., Etzkorn, C., Barbas, C.F. III, Segal, D.J., Horton, N.C. Crystallographic

analysis of Aart, a designed six-finger zinc finger peptide, bound to DNA.

Acta Crystallogr. F61:573, 2005.

Gräslund, T., Li, X., Popkov, M., Barbas, C.F. III. Exploring strategies for the

design of artificial transcription factors: targeting sites proximal to known regulatory

regions for the induction of γ-globin expression and the treatment of sickle cell disease.

J. Biol. Chem. 280:3707, 2005.

Haba, K., Popkov, M., Shamis, M., Lerner, R.A., Barbas, C.F. III, Shabat, D. Single-triggered

trimeric prodrugs, Angew. Chem. Int. Ed. 44:716, 2005.

Jendreyko, N., Popkov, M., Rader, C., Barbas, C.F. III. Phenotypic knockout of

VEGF-R2 and Tie-2 with an intradiabody reduces tumor growth and angiogenesis

in vivo. Proc. Natl. Acad. Sci. U. S. A. 102:8293, 2005.

Li, L.-S., Rader, C., Matsushita, M., Das, S., Barbas, C.F, III, Lerner, R.A., Sinha,

S.C. Chemical adaptor immunotherapy: design, synthesis, and evaluation of novel

integrin-targeting devices. J. Med. Chem. 47:5630, 2004.

Magnenat, L., Blancafort, P., Barbas, C.F. III. In vivo selection of combinatorial libraries

and designed affinity maturation of polydactyl zinc finger transcription factors for

ICAM-1 provides new insights into gene regulation. J. Mol. Biol. 341:635, 2004.

Mase, N., Thayumanavan, R., Tanaka, F., Barbas, C.F. III. Direct asymmetric organocatalytic

Michael reactions of α,α-disubstituted aldehydes with β-nitrostyrenes for

the synthesis of quaternary carbon-containing products. Org. Lett. 6:2527, 2004.

Notz, W., Tanaka, F., Barbas, C.F. III. Enamine-based organocatalysis with proline

and diamines: the development of direct catalytic asymmetric aldol, Mannich,

Michael, and Diels-Alder reactions. Acc. Chem. Res. 37:580, 2004.

Notz, W., Watanabe, S., Chowdari, N.S., Zhong, G., Betancort, J.M., Tanaka, F.,

Barbas, C.F. III. The scope of the direct proline-catalyzed asymmetric addition of

ketones to imines. Adv. Synth. Catal. 346:1131, 2004.

Popkov, M., Jendreyko, N., McGavern, D.B., Rader, C., Barbas, C.F. III. Targeting

tumor angiogenesis with adenovirus-delivered anti-Tie-2 intrabody. Cancer Res.

65:972, 2005.

Popkov, M., Rader, C., Barbas, C.F. III. Isolation of human prostate cancer cell reactive

antibodies using phage display technology. J. Immunol. Methods 291:137, 2004.

Ramachary, D.B., Barbas, C.F. III. Direct amino acid-catalyzed asymmetric desymmetrization

of meso-compounds: tandem aminoxylation/O-N bond heterolysis reactions.

Org. Lett. 7:1577, 2005.

Ramachary, D.B., Barbas, C.F. III. Towards organo-click chemistry: development

of organocatalytic multicomponent reactions through combinations of aldol, Wittig,

Knoevenagel, Michael, Diels-Alder and Huisgen cycloaddition reactions. Chemistry

10:5323, 2004.

Sinha, S.C., Li, l.-S., Watanabe, S., Kaltgrad, E., Tanaka, F., Rader, C., Lerner,

R.A., Barbas, C.F. III. Aldolase antibody activation of prodrugs of potent aldehydecontaining

cytotoxics for selective chemotherapy. Chemistry 10:5467, 2004.

Steiner, D.D., Mase, N., Barbas, C.F. III. Direct asymmetric α-fluorination of aldehydes.

Angew. Chem. Int. Ed. 44:3706, 2005.

Suri, J.T., Ramachary, D.B., Barbas, C.F. III. Mimicking dihydroxy acetone phosphate-utilizing

aldolases through organocatalysis: a facile route to carbohydrates

and aminosugars. Org. Lett. 7:1383, 2005.

Tanaka, F., Barbas, C.F. III. Enamine-based reactions using organocatalysts: from

aldolase antibodies to small amino acid and amine catalysts. J. Synth. Org. Chem.

Jpn., in press.

Tanaka, F., Barbas, C.F. III. Organocatalytic approaches to enantioenriched β-amino

acids. In: Enantioselective Synthesis of β-Amino Acids, 2nd ed. Juaristi, E., Soloshonok,

V. (Eds.). Wiley-VCH, New York, 2004, p. 195.

Tanaka, F., Barbas, C.F. III. Reactive immunization: a unique approach to aldolase antibodies.

In: Catalytic Antibodies. Keinan, E. (Ed.). Wiley-VCH, New York, 2004, p. 304.

Tanaka, F., Flores, F., Kubitz, D., Lerner, R.A., Barbas, C.F. III. Antibody-catalyzed

aminolysis of a chloropyrimidine derivative. Chem. Commun. (Camb.) 1242,

2004, Issue 10.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Tanaka, F., Mase, N., Barbas, C.F. III. Determination of cysteine concentration by

fluorescence increase: reaction of cysteine with a fluorogenic aldehyde. Chem.

Commun. (Camb.) 1762, 2004, Issue 15.

Thayumanavan, R., Tanaka, F., Barbas, C.F. III. Direct organocatalytic asymmetric

aldol reactions of α-amino aldehydes: expedient synthesis of highly enantiomerically

enriched anti-β-hydroxy-α-amino acids. Org. Lett. 6:3541, 2004.

Zhong, G., Fan, J., Barbas, C.F. III. Amino alcohol catalyzed direct asymmetric

aldol reactions: enantioselective synthesis of anti-α-fluoro-β-hydroxy ketones. Tetrahedron

Lett. 45:5681, 2004.

Zhu, X., Tanaka, F., Hu, Y., Heine, A., Fuller, R., Zhong, G., Olson, A.J., Lerner,

R.A., Barbas, C.F. III, Wilson, I.A. The origin of enantioselectivity in aldolase antibodies:

crystal structure, site-directed mutagenesis, and computational analysis. J.

Mol. Biol. 343:1269, 2004.

Synthetic Enzymes, Catalytic

Antibodies, Ozone Scavengers,

Organic Synthesis, and

Biomolecular Computing

E. Keinan, C.H. Lo, H. Han, S. Sasmal, S. Ledoux,

N. Metanis, G. Sklute, E. Kossoy, M. Soreni, D. Vebenov,

R. Piran, M. Sinha, A. Alt, I. Ben-Shir, R. Girshfeld, S. Yogev

We focus on synthetically modified enzymes,

antibody-catalyzed reactions, anticancer and

antiasthma agents, and biomolecular computation,

as illustrated in the following examples.

SYNTHETIC ENZYMES

MOLECULAR BIOLOGY 2005 213

Efforts to generate new enzymatic activities from

existing protein scaffolds may not only provide biotechnologically

useful catalysts but also lead to better

understanding of the natural process of evolution. We

profoundly changed the catalytic activity and mechanism

of the enzyme 4-oxalocrotonate tautomerase by

means of rationally designed synthetic mutations. For

example, a single amino acid substitution that corresponds

to a mutation in a single base pair led to a

dramatic change in the catalytic activity. Although the

wild-type enzyme catalyzes only the tautomerization of

4-oxalocrotonate, the mutant P1A catalyzes both the

original tautomerization reaction via a general acid-base

mechanism and the decarboxylation of oxaloacetate via

a nucleophilic mechanism. The observation that a single

catalytic group in an enzyme can catalyze 2 reactions

by 2 different mechanisms supports the hypothesis

that enzyme evolution is a continuum in which a new

catalytic mechanism is gained while the parent activity

declines gradually through small changes in the amino

acid sequence of the primordial enzyme.


214 MOLECULAR BIOLOGY 2005

We also showed that the electrostatic manipulation

of an enzyme’s active site can alter the substrate specificity

of the enzyme in a predictable way. We replaced

1, 2, or all 3 active-site arginine residues with citrulline

analogs to maintain the steric features of the active

site of 4-oxalocrotonate tautomerase while changing

its electronic properties. These synthetic changes

revealed that the wild-type enzyme binds the natural

substrate predominantly through electrostatic interactions.

This and other mechanistic insights led to the

design of a modified enzyme that was specific for a

new substrate that had different electrostatic properties

and that bound the enzyme via hydrogen-bonding

complementarity rather than electrostatic interactions.

The synthetic analog of the natural 4-oxalocrotonate

tautomerase was a poor catalyst of the natural 4-oxalocrotonate

substrate but an efficient catalyst for a ketoamide

substrate. This research on synthetic enzymes

is being done in collaboration with P.E. Dawson, Department

of Cell Biology.

CATALYTIC ANTIBODIES

Although the solution photochemical reaction of

the ketone 1 (in Fig. 1) yields only the cleavage products

2 and 3, in the presence of 20F10, an antibody

to 5a and 5b, this Norrish type II reaction results in the

selective formation of cis-cyclobutanol (compound 4

in Fig. 1). Furthermore, the fact that compound 4,

which consists of 2 asymmetric centers, is obtained

as a single diastereomer makes this photoproduct a

valuable building block for the synthesis of natural

products. Another reaction that is exclusively catalyzed

by 20F10 is the photochemical formation of cyclopropanol

products.

Fig. 1. The photochemical Norrish type II reaction of ketone 1

produces in solution the cleavage products 2 and 3. Antibody

20F10, which was elicited against a mixture of 5a and 5b, catalyzes

enantioselective formation of cis-cyclobutanol (4).

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

An aldolase antibody, 24H6, obtained from immunization

with large diketone haptens has an active-site

lysine residue with a perturbed pK a of 7.0. This antibody

catalyzes both the aldol addition and the retrograde

aldol fragmentation with a broad range of substrates that

differ structurally from the hapten. This observation

suggests that in reactive immunization with 1,3-diketones,

the hapten structure governs the chemistry but

not the overall organization of the active site. Antibody

24H6 also catalyzes the oxidation of α-hydroxyketones

to α-diketones. The deuterium exchange at the α position

of many ketones and aldehydes is also efficiently

catalyzed by aldolase antibodies 38C2 and 24H6. All

reactions were carried out in deuterium oxide under

neutral conditions and showed regioselectivity, chemoselectivity,

and high catalytic rates.

OZONE SCAVENGERS AND ANTIASTHMA ACTIVITY

A new hypothesis we proposed for the mechanism

of asthmatic inflammation has led to an ozone-scavenging

compound that prevents bronchial obstruction

in rats with asthma. Previously, scientists at Scripps

Research discovered that ozone can be generated not

only via the antibody-mediated water oxidation pathway

but also by antibody-coated activated white blood

cells during inflammatory processes. This finding led

us to speculate that the pulmonary inflammation in

asthma might be caused by ozone production by white

blood cells in lungs and that inhalation of electron-rich

olefins, which are known ozone scavengers, might have

antiasthmatic effects. In experiments in rats, inhalation

of such a compound, limonene, caused a significant

improvement in asthmatic symptoms. These

results could have consequences in the management

of asthma.

ORGANIC SYNTHESIS

Annonaceous acetogenins, particularly those with

adjacent bis-tetrahydrofuran rings, have remarkable

cytotoxic, antitumor, antimalarial, immunosuppressive,

pesticidal, and antifeedant activities. More than 350

different acetogenins have been isolated from only 35

of 2300 plants of the family Annonaceae. We developed

synthetic approaches that can be used to generate

chemical libraries of stereoisomeric acetogenins.

These efforts resulted in the total synthesis of several

naturally occurring acetogenins, including asimicin,

bullatacin, trilobacin, rolliniastatin, solamin, reticulatacin,

rollidecins C and D, goniocin, cyclogoniodenin,

and mucocin, and many nonnatural stereoisomers. A

substituted photoactive derivative of asimicin has been


prepared for photoaffinity labeling of the target protein

subunit in the mitochondrial complex I. This research

is being done in collaboration with S.C. Sinha, Department

of Molecular Biology.

BIOMOLECULAR COMPUTING DEVICES

Four years ago we described the first nanoscale,

programmable finite automaton with 2 symbols and 2

states that computed autonomously. All of the components

of the device, including hardware, software, input,

and output, were biomolecules mixed together in solution.

The hardware consisted of a restriction nuclease

and a ligase; the software (transition rules) and the

input were double-stranded DNA oligomers (Fig. 2).

Fig. 2. A biomolecular computing machine made of molecules.

The hardware consists of a restriction nuclease and a ligase; the

input, transition molecules (software), and detection molecules are

all made of double-stranded DNA.

Computation was carried out by processing the input

molecule via repetitive cycles of restriction, hybridization,

and ligation reactions to produce a final-state output

in the form of a double-stranded DNA molecule.

Currently, we are taking the concept of molecular computing

a step further and are constructing computing

devices in which the computation output is a specific

biological function rather than a specific molecule.

Most recently, we markedly increased the levels of

complexity and mathematical power of these automata

by the design of a 3-state–3-symbol automaton, thus

increasing the number of syntactically distinct programs

from 765 to 1 billion. We have further amplified the

applicability of this design by using surface-anchored

input molecules and surface plasmon resonance technology

to monitor the computation steps in real time.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

This technology allowed parallel computation and automatic,

real-time detection with DNA chips that carry

multiple input molecules and can be used as pixel arrays

for image encryption.

PUBLICATIONS

Dubnikova, F., Kosloff, R., Almog, J., Zeiri, Y., Boese, R., Itzhaky, H., Alt, A.,

Keinan, E. Decomposition of triacetone triperoxide is an entropic explosion. J. Am.

Chem. Soc. 127:1146, 2005.

Keinan, E., Alt, A., Amir, G., Bentur, L., Bibi, H., Shoseyov, D. Natural ozone

scavenger prevents asthma in sensitized rats. Bioorg. Med. Chem. 13:557, 2005.

Metanis, N., Keinan, E., Dawson, P.E. A designed synthetic analogue of 4-OT is

specific for a non-natural substrate. J. Am. Chem. Soc. 127:5862, 2005.

Saphier, S., Hu, Y., Sinha, S.C., Houk, K.N., Keinan, E. The origin of selectivity in the

antibody 20F10-catalyzed Yang cyclization. J. Am. Chem. Soc. 127:132, 2005.

Soreni, M., Yogev, S., Kossoy, E., Shoham, Y., Keinan, E. Parallel biomolecular

computation on surfaces with advanced finite automata. J. Am. Chem. Soc.

127:3935, 2005.

Antibody Catalysis and

Organic Synthesis

S.C. Sinha, R.A. Lerner, S. Das, S. Abraham, F. Guo,

Z. Chen

MOLECULAR BIOLOGY 2005 215

Our main research interests are antibody catalysis

and the applications of antibody catalysts

in organic synthesis, prodrug activation, and

the development of cell-targeting antibody constructs.

In addition, we also focus on synthetic and medicinal

chemistry, including the total synthesis of biologically

important natural products and synthetic compounds

and their analogs and new methods of synthesis.

ANTIBODY CATALYSIS AND ITS APPLICATIONS

Aldolase antibodies 38C2, 84G3, and 93F3 produced

by the reactive immunization technique are

highly useful in synthetic organic chemistry, as indicated

by their application in the syntheses of a number

of natural products, including epothilones. These

antibodies catalyze both aldol and retro-aldol reactions

and yield products with high enantioselectivities.

The high catalytic rate of the retro-aldol reaction makes

the antibodies useful in prodrug therapy.

In prodrug therapy, an enzyme or a catalytic antibody

is used to activate a nontoxic prodrug at a targeted

site, thereby producing a cytotoxic drug. We are developing

prodrugs of cytotoxic molecules, including paclitaxel,

doxorubicin analogs, enediynes, CBI analogs, and

epothilones, that can be activated efficiently by aldolase

antibodies. In particular, we prepared and evaluated


216 MOLECULAR BIOLOGY 2005

several prodrugs of the analogs of dynemicin B and doxorubicin.

The prodrugs of dynemicin analogs are activated

by using antibody 38C2; those of doxorubicin analogs,

by 93F3 (Fig. 1). On the basis of these studies, we are

developing new linkers for the prodrugs so that activation

of the prodrugs can be selectively achieved at

high catalytic rate.

Fig. 1. Structure of the prodrugs of doxorubicin (DOX) analogs.

Using antibody 38C2, we also developed antagonist-38C2

conjugates. The conjugates bound efficiently

to cells expressing the integrins α v β 3 and α v β 5 . The

conjugates have several advantages, including prolongation

of half-life of the antagonist and in vivo assembly

of the conjugate. On the basis of our initial studies,

in collaboration with C.F. Barbas, Department of Molecular

Biology, we synthesized a series of antagonist-38C2

conjugates and evaluated them by using breast cancer

cell lines that express the integrins α v β 3 and α v β 5 . Several

conjugates (Fig. 2) bound to these cell lines with

high affinity. Our findings, which were supported by

molecular docking studies, provided preliminary information

on how the compounds should be derivatized.

Fig. 2. Structure of the compounds that target the integrins α v β 3

and α v β 5 for conjugation with 38C2.

SYNTHESIS OF NATURAL PRODUCTS AND THEIR

ANALOGS

In the past year, we focused on the synthesis of

naturally occurring macrocyclic molecules, sorangiolides

A and B, and the library of bis-tetrahydrofuran annonaceous

acetogenins. Sorangiolides (Fig. 3) are weakly

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Fig. 3. Structure of sorangiolides A and B (top) and a general

structure of bis-tetrahydrofuran annonaceous acetogenins (bottom).

active against gram-positive bacteria. Our goal is to

synthesize analogs of sorangiolides that are highly active.

The bis-tetrahydrofuran acetogenins are among the

most active cancer agents and are toxic to a number of

human cancer cell lines at much lower concentrations

than doxorubicin is. In collaboration with E. Keinan,

Department of Molecular Biology, we synthesized an

analog of asimicin, an annonaceous acetogenin, for

photoaffinity labeling of the corresponding receptor. In

other studies, we developed a bidirectional approach for

the synthesis of all 64 diastereomers of the adjacent

bis-tetrahydrofuran acetogenins (Fig. 3). Starting with

8 diene lactones, we synthesized 36 bifunctional adjacent

bis-tetrahydrofuran lactones by using 5 key reactions:

(1) monooxidative or bis-oxidative cyclization

mediated by rhenium(VII) oxides, (2) Shi monoasymmetric

or bis-asymmetric epoxidation, (3) Sharpless

asymmetric dihydroxylation, (4) Williamson-type etherification,

and (5) Mitsunobu inversion. Further studies

are in progress.

PUBLICATIONS

Li, L.-S., Rader, C., Matsushita, M., Das, S., Barbas, C.F. III, Lerner, R.A., Sinha,

S.C. Chemical-adaptor immunotherapy: design, synthesis and evaluation of novel

integrin-targeting devices. J. Med. Chem. 47:5630, 2004.

Saphier, S., Hu, Y., Sinha, S.C., Houk, K.N., Keinan, E. Origin of selectivity in the

antibody 20F10-catalyzed Yang cyclization. J. Am. Chem. Soc. 127:132, 2005.

Sinha, S.C., Li, L.-S., Watanabe, S.-I., Kaltgrad, E., Tanaka, F., Rader, C., Lerner,

R.A., Barbas, C.F. III. Aldolase antibody activation of prodrugs of potent aldehydecontaining

cytotoxics for selective chemotherapy. Chemistry 10:5467, 2004.


Structure, Function, and

Applications of Virus Particles

J.E. Johnson, L. Basumallic, A. Chatterji, W. Fernandez-

Ochoa, L. Gan, I. Gertsman, R. Khayat, J. Lanman, K. Lee,

T. Matsui, P. Natarajan, A. Odegard, J. Speir, L. Tang,

H. Walukiewicz, E. Wu

We investigate model virus systems that provide

insights for understanding assembly, maturation,

entry, localization, and replication of

nonenveloped viruses. We also have developed viruses

as reagents for applications in nanotechnology, chemistry,

and biology. We investigate viruses that infect bacteria,

insects, yeast, plants, and, recently, the extreme

thermophile Sulfolobus. These viruses have genomes

of single-stranded RNA, double-stranded RNA, and double-stranded

DNA. In many instances, we use viruslike

particles that do not contain infectious genomes.

We use a variety of physical methods to investigate

structure-function relationships, including single-crystal

and static and time-resolved solution x-ray diffraction,

electron cryomicroscopy and image reconstruction, mass

spectrometry, structure-based computational analyses,

and methods associated with thermodynamic characterization

of virus particles and their transitions. Biological

methods we use include genetic engineering of viral

genes and their expression in Escherichia coli, mammalian

cells, insect cells, and yeast and the characterization

of these gene products by the physical methods

mentioned previously. For cytologic studies of viral

entry and infection, we use fluorescence and electron

microscopy and particles assembled in heterologous

expression systems. Our studies depend on extensive

consultations and collaborations with others at Scripps

Research, including groups led by C.L. Brooks, D.A.

Case, B. Carragher, M.G. Finn, M.R. Ghadiri, T. Lin,

M. Manchester, D.R. Millar, R.A. Milligan, C. Potter,

V. Reddy, A. Schneemann, G. Siuzdak, K.F. Sullivan,

J.R. Williamson, and M.J. Yeager, and a variety of groups

outside of Scripps.

DOUBLE-STRANDED DNA VIRUSES

HK97 is a double-stranded DNA bacterial virus

similar to phage λ. It undergoes a remarkable morphogenesis

in its assembly and maturation, and this process

can be recapitulated in vitro. We determined the

atomic resolution structure of the 650-Å mature head

II particle and discovered the mechanism used to concatenate

the subunits of the particle into a chain-mail

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 217

fabric similar to that seen in the armor of medieval

knights. We created a model of the procapsid on the

basis of the 5-Å electron cryomicroscopy structure in

which the coordinates from the head II particle were

readily fitted. Recently, we used single-value decomposition

analysis of time-resolved solution x-ray scattering

data and single-molecule fluorescence to show

that the initial maturation of prohead II (~470 Å in

diameter) to expansion intermediate I (546 Å in diameter)

occurs as a highly cooperative, stochastic event

with no significantly populated intermediates and takes

less than 1 second for an individual particle.

Bacteriophage P22 is the prototype of the Podoviridae,

which are characterized by a T = 7 capsid with

a short tail structure incorporated into a unique 5-fold

vertex. We previously determined the icosahedrally averaged

structure of the capsid at 20-Å resolution, the 10-Å

structure of the connector protein, and the 20-Å structure

of the tail machine. Recently, we did a reconstruction

of the virus without imposing symmetry, enabling

us to visualize the detailed relationship of all these

components (Fig. 1).

Fig. 1. Electron cryomicroscopy reconstruction of the bacteriophage

P22. The reconstruction was done with 1800 particles and

no applications of symmetry. The reconstructed density required

first generating an icosahedrally averaged electron density that ignored

the tail assembly and then inserting the tail assembly (determined

as a separate reconstruction) into the icosahedral density to create

a tailed phage model. The model was then back projected in all icosahedral

orientations onto each individual particle, and the orientation

that gave the highest correlation coefficient (i.e., aligned the

tails) was used to reconstruct the final density. Note that without

any application of symmetry, both the tail machine and the T = 7

surface lattice are clearly defined in the reconstructed density.


218 MOLECULAR BIOLOGY 2005

Sulfolobus turreted icosahedral virus is an archaeal

virus isolated from Sulfolobus, which grows in the

acidic hot sulfur springs (pH 2–4, 72°C–92°C) in Yellowstone

National Park. An electron cryomicroscopy

reconstruction of the virus showed that the capsid has

pseudo T = 31 quasi symmetry and is 1000 Å in diameter,

including the pentons. We solved the x-ray structure

of the major capsid protein of the virus and revealed

a fold nearly identical to the major capsid proteins of

the eukaryotic adenoviruses; PBCV-1, a virus that infects

fresh water algae; and PRD-1, a virus that infects bacteria.

These findings indicate a virus phylogeny that spans

the 3 domains of life (Eucarya, Bacteria, and Archaea)

and suggests that these viruses may be related to a

virus that preceded the division of life into 3 domains

more than 3 billion years ago.

SINGLE-STRANDED RNA VIRUSES

Flock House virus is a single-stranded RNA virus

that infects Drosophila. We are studying viral entry

and early expression and assembly of the capsid protein.

Recently, studies on viral entry indicated the

presence of an “eluted” particle early in infection that

has initiated its disassembly program but is then eluted

back into the medium. We did a phenotypic characterization

of the particles, and we are using electron cryomicroscopy

to study them. For studies on the expression

and assembly of the capsid protein, we are using tags

genetically inserted in the capsid protein that allow the

freshly made proteins to be optically visualized with a

fluorophore and in the electron microscope with photoconversion

of the fluorophore.

Tetraviruses are single-stranded RNA viruses that

infect Lepidoptera. Expression of the capsid protein in

the baculovirus system leads to spontaneous assembly

of viruslike particles that we can investigate in vitro. The

particles exist as procapsids (480 Å) at pH 7 and as

capsids (410 Å) at pH 5. We used limited proteolysis

and mass spectrometry to investigate the driving force

of the transition, the mechanism of an autocatalytic

cleavage, and the dynamic features of both forms.

Cowpea mosaic virus is a 30-nM reagent that we use

for chemistry and nanotechnology. In collaboration with

T. Lin, Department of Molecular Biology, we generated

and produced a large variety of viable mutations of the

virus in gram quantities for nanopatterning, molecular

electronic scaffolds, and platforms for novel chemistry.

PUBLICATIONS

Blum, A.S., Soto, C.M. Wilson, C.D., Brower, T.L., Pollack, S.K., Schull, T.L.,

Chatterji, A., Lin, T., Johnson, J.E., Amsinck, C., Franzon, P., Shashidhar, R.,

Ratna, B.R. An engineered virus as a scaffold for three-dimensional self-assembly

on the nanoscale. Small 1:702, 2005.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Blum, A.S., Soto, C.M., Wilson, C.D., Cole, J.D., Kim, M., Gnade, B., Chatterji, A.,

Ochoa, W.F., Lin, T., Johnson, J., Ratna, B.R. Cowpea mosaic virus as a scaffold

for 3-D patterning of gold nanoparticles. Nano Lett. 4:867, 2004.

Bothner, B., Taylor, D., Jun, B., Lee, K.K., Siuzdak, G., Schultz, C.P., Johnson,

J.E. Maturation of a tetravirus capsid alters the dynamic properties and creates a

metastable complex. Virology 334:17, 2005.

Chatterji, A., Ochoa, W.F., Paine, M., Ratna, B.R., Johnson, J.E., Lin, T. New

addresses on an addressable virus nanoblock; uniquely reactive Lys residues on

cowpea mosaic virus. Chem. Biol. 11:855, 2004.

Chatterji, A., Ochoa, W.F., Ueno, T., Lin, T., Johnson, J.E. A virus-based nanoblock

with tunable electrostatic properties. Nano Lett. 5:597, 2005.

Falkner, J.C., Turner, M.E., Bosworth, J.K., Trentler, T.J., Johnson, J.E., Lin, T.,

Colvin, V.L. Virus crystals as nanocomposite scaffolds. J. Am. Chem. Soc.

127:5274, 2005.

Girard, E., Kahn, R., Mezouar, M., Dhaussy, A.C., Lin, T., Johnson, J.E., Fourme, R.

The first crystal structure of a complex macromolecular assembly under high pressure:

CpMV at 330 MPa. Biophys. J. 88:3562, 2005.

Johnson, K.N., Tang, L., Johnson, J.E., Ball, L.A. Heterologous RNA encapsidated

in Pariacoto virus-like particles forms a dodecahedral cage similar to genomic RNA

in wild-type virions. J. Virol. 78:11371, 2004.

Lin, T., Lomonossoff, G.P., Johnson, J.E. Structure-based engineering of an icosahedral

virus for nanomedicine and nanotechnology. In: Nanotechnology in Biology

and Medicine: Methods, Devices, and Applications. Vo-Dinh, T. (Ed.). CRC Press,

Boca Raton, FL, in press.

Lin, T., Schildkamp, W., Brister, K., Doerschuk, P.C., Somayazulu, M., Mao,

H.K., Johnson, J.E. The mechanism of high pressure induced ordering in a macromolecular

crystal. Acta Crystallogr. D Biol. Crystallogr. 61:737, 2005.

Medintz, I., Mattoussi, H., Sapsford, K., Chatterji, A., Johnson, J.E. Decoration of

discretely immobilized cowpea mosaic virus with luminescent quantum dots. Langmuir,

in press.

Natarajan, P., Lander, G., Shepherd, C., Reddy, V., Brooks, C.L. III, Johnson, J.E.

Virus Particle Explorer (VIPER), a Web-based repository of virus structural data and

derived information. Nat. Microbiol. Rev., in press.

Reddy, V., Schneemann, A., Johnson, J.E. Nodavirus endopeptidase. In: Handbook

of Proteolytic Enzymes, 2nd ed. Barret, A.J., Rawlings, N.D., Woessner, J.F.

(Eds.). Academic Press, San Diego, 2004, Vol. 2, p. 198.

Reddy, V.S., Natarajan, P., Lander, G., Qu, C., Brooks, C.L. III, Johnson, J.E.

Virus Particle Explorer (VIPER): a repository of virus capsid structures. In: Conformational

Proteomics of Macromolecular Architecture: Approaching the Structure of

Large Molecular Assemblies and Their Mechanisms of Action. Cheng, R.H., Hammar,

L. (Eds.). World Scientific, River Edge, NJ, 2004, p. 403.

Schwarcz, W.D., Barroso, S.P., Gomes, A.M., Johnson, J.E., Schneemann, A.,

Oliveira, A.C., Silva, J.L. Virus stability and protein-nucleic acid interaction as

studied by high-pressure effects on nodaviruses. Cell. Mol. Biol. (Noisy-le-grand)

50:419, 2004.

Strable, E., Johnson, J.E., Finn, M.G. Natural supramolecular building blocks:

icosahedral virus particles organized by attached oligonucleotides. Nano Lett.

4:1385, 2004.

Tang, J., Naitow, H., Gardner, N.A., Kolesar, A., Tang, L., Wickner, R.B., Johnson,

J.E. The structural basis of recognition and removal of cellular mRNA 7-methyl G

“caps” by a viral capsid protein: a unique viral response to host defense. J. Mol.

Recognit. 18:158, 2005.

Tang, L., Marion, W.R., Cingolani, G., Prevelige, P.E., Johnson, J.E. The three-dimensional

structure of the bacteriophage P22 tail machine. EMBO J. 24:2087, 2005.

Taylor, D.J., Johnson, J.E. Folding and particle assembly are disrupted by singlepoint

mutations near the autocatalytic cleavage site of nudaurelia capensis 4 virus

capsid protein. Protein Sci. 14:401, 2005.

Taylor, D.J., Speir, J., Reddy, V., Cingolani, G., Pringle, F., Ball, L.A., Johnson,

J.E. Preliminary x-ray characterization of authentic providence virus and attempts

to express its coat protein gene in recombinant baculovirus. Arch. Virol., in press.


An Icosahedral Scaffold for

Biophysical Studies and

Nanomanufacturing

T. Lin, J.E. Johnson, A. Chatterji, W.F. Ochoa, A. Stone,

T. Ueno

Cowpea mosaic virus (CPMV) is an icosahedral

plant virus with a diameter of 30 nm. Because

of its exceptional stability, high yield, ease of

production, structural information to the level of atomic

definition, and accessible genetic programmability, the

virus has been used as a model system for biophysical

studies and has been engineered for applications in

biotechnology and nanotechnology.

ASSEMBLY OF NANOMATERIALS ON AN

ICOSAHEDRAL SCAFFOLD

A quintessential tenet of nanotechnology is the selfassembly

of components at nanometer scale to form

devices. Although small molecules with novel electronic

properties can be synthesized, it is generally difficult

to get functional connectivity among the different components

in designed patterns. In contrast, because of

their versatility, programmability through genetic engineering,

and propensity to form arrays, biological macromolecules

are more amenable for self-assembly either

as devices for direct use or as scaffolds for patterning

small molecules. We showed that CPMV can be used

as a template for nanochemistry by introducing unique

cysteine residues and exploiting the native lysine residues.

In collaboration with B.R. Ratna, Naval Research

Laboratory, Washington, D.C., we used the viral capsid

as a nano circuit board and the reactive groups as

anchoring points for the assembly of electronic molecules,

oligophenylene-vinylene and others. The establishment

of the molecular network was demonstrated

by measuring electronic conductance via scanning tunnel

microscopy.

HIGH-PRESSURE CRYSTALLOGRAPHY

Using high pressure, we markedly improved the diffraction

from the cubic crystals of CPMV from about 4-Å

to 2.1-Å resolution. If this use of pressure is generally

applicable, it can have a marked effect on structural biology.

To this end, we carried out mechanistic studies of

the pressure-induced rectification of crystal imperfection.

Two types of cubic crystals were assigned to either

an I23 or a P23 space group. The 2 types had the same

rhombic dodecahedral morphology at atmospheric pres-

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

sure. The crystals assigned to the I23 space group diffracted

x-rays to higher resolution than did those

assigned to the P23 space group. The assignment of

the P23 space group was due to the presence of reflections

with indices h + k + l = (2n + 1) (odd reflections),

which are forbidden in the I23 space group.

Analysis of the odd reflections from the P23 crystals

at atmospheric pressure indicated that they originated

from a rotational disorder in the the I23 crystals. The

odd reflections were eliminated by applying 3.5 kbar

of pressure, which transformed the crystals from the

apparently primitive cell to the body-centered I23 cell,

with dramatic improvement in diffraction.

PUBLICATIONS

Blum, S.A., Soto, C.M., Wilson, C.D., Brower, T.L., Pollack, S.K., Schull, T.L.,

Chatterji, A., Lin, T., Johnson, J.E., Amsinck, C., Franson, P., Shashidhar, R.,

Ratna, B.R. An engineered virus as a scaffold for three-dimensional self-assembly

on the nanoscale. Small 1:702, 2005.

Chatterji, A., Ochoa, W., Shamieh, L., Salakian, S.P., Wong, S.M., Clinton, G.,

Ghosh, P., Lin, T., Johnson, J.E. Chemical conjugation of heterologous proteins on

the surface of cowpea mosaic virus. Bioconjug. Chem. 15:807, 2004.

Chatterji, A., Ochoa, W.F., Paine, M., Ratna, B.R., Johnson, J.E., Lin, T. New

addresses on an addressable virus nanoblock: uniquely reactive Lys residues on

cowpea mosaic virus. Chem. Biol. 11:855, 2004.

Chatterji, A., Ochoa, W.F., Ueno, T., Lin, T., Johnson, J.E. A virus-based

nanoblock with tunable electrostatic properties. Nano Lett. 5:597, 2005.

Falkner, J.C., Turner, M.E., Bosworth, J.K., Trentler, T.J., Johnson, J.E., Lin, T.,

Colvin, V.L. Virus crystals as nanocomposite scaffolds. J. Am. Chem. Soc.

127:5274, 2005.

Girard, E., Kahn, R., Mezouar, M., Dhaussy, A.-C., Lin, T., Johnson J.E., Fourme, R.

The first crystal structure of a complex macromolecular assembly under high pressure:

CpMV at 330 MPa. Biophys. J. 88:3562, 2005.

Lin, T., Schildkamp, W., Brister, K., Doerschuk, P.C., Somayazulu, M., Mao H.,

Johnson, J.E. The mechanism of high-pressure-induced ordering in a macromolecular

crystal. Acta Crystallogr. D Biol. Crystallogr. 61:737, 2005.

Design and Informatics in

Structural Virology

V.S. Reddy, C.M. Shepherd, C. Hsu, S. Kumar, R. Mannige,

I. Borelli, C.L. Brooks III, J.E. Johnson, M. Manchester,

A. Schneemann

MOLECULAR BIOLOGY 2005 219

We are interested in understanding the structural

underpinnings and requirements for

formation of viral capsids and in designing

novel protein shells that polyvalently display molecules

of interest. To this end, we use structural, computational,

informatics, and genetic methods.

Viruses are highly evolved macromolecular machines

that perform a variety of functions during their life cycle,


220 MOLECULAR BIOLOGY 2005

including selective packaging of the genome, selfassembly,

binding to host cells, and delivery of the

genome to the targeted cells. Simple viruses, such as

nonenveloped viruses, form closed protein shells or

capsids of uniform size and character by the self-association

of structural and functional components: proteins

and the nucleic acid genome. Hence, these viruses are

useful for structural and functional analyses.

To understand the requirements for formation of

the closed protein shell in viral capsids in terms of

structure and interactions, we established a repository

of structurally characterized viral capsids in a relational

database format, namely the Viper Particle Explorer

(http://viperdb.scripps.edu). At the database, we use

computational methods to analyze these protein shells

in terms of protein-protein interactions: contacting residue

pairs, association energies, individual residue contributions,

and surface characteristics. To facilitate these

studies, we are developing structural tools for analysis

of viral structures as part of the Multiscale Modeling

Tools for Structural Biology, the National Institutes of

Health research resource headed by C.L. Brooks, Department

of Molecular Biology. The structural and taxonomic

data and the derived results are stored in a MySQL

database for ease of querying and comparing the properties

of interest within and across families of viruses. Furthermore,

using the structural similarity that occurs

within a virus family, we are building homology models

for the uncharacterized members of virus families. These

models will be useful for molecular virologists investigating

structural and functional relationships in viruses.

To generate novel reagents, such as vaccines and

antitoxins against cytotoxins such as ricin and pathogens

in general, we are expressing decoys of pathogenic

molecules on the surfaces of viral capsids. Currently,

tomato bushy stunt virus–like capsids are the display

platform of choice; the platform consists of multiple

copies of a 2-domain capsid protein subunit with the

C-terminal P-domain exposed on the surface. Such a

unique subunit structure is useful for attaching peptides

or proteins of interest at the end of the C terminus of

the capsid protein or for replacing the P-domain with the

proteins of interest rather than inserting them in a loop.

PUBLICATIONS

Reddy, V.S., Johnson J.E. Structure-derived insights into virus assembly. Adv. Virus

Res. 64C:45, 2005.

Reddy, V.S., Schneemann, A., Johnson, J.E. Nodavirus endopeptidase. In: Handbook

of Proteolytic Enzymes, 2nd ed. Barrett, A., Rawlings, N.D., Woessner, J.F.

(Eds.). Academic Press, San Diego, 2004, p. 197.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Shepherd, C.M., Reddy, V.S. Extent of protein-protein interactions and quasi-equivalence

in viral capsids. Proteins 58:472, 2005.

Biology and Applications of

Capsids of Icosahedral Viruses

A. Schneemann, B. Groschel, J. Lee, D. Manayani,

M. Siladi, P.A. Venter

Coat proteins of nonenveloped, icosahedral viruses

perform multiple functions during the course of

viral infection, including capsid assembly, specific

encapsidation of the viral genome, binding to a

cellular receptor, and uncoating. In some viruses, a

single type of protein is sufficient to carry out these

functions; we are interested in the determinants that

endow a polypeptide chain with such versatility. We

seek to harness this versatility for novel applications

of viruses in biotechnology and nanotechnology.

We focus on a structurally and genetically wellcharacterized

virus family, the T = 3 nodaviruses.

Nodaviruses are composed of 180 copies of a single

coat protein and 2 strands of positive-sense RNA. Currently,

we are elucidating the mechanism by which the

2 genomic RNAs are packaged into a single virion. Our

long-term goal is to develop nodaviruses as RNA packaging

and delivery vectors. Our data indicate that the

2 viral RNAs are recognized separately, but it is not

yet known whether packaging occurs sequentially and

whether one or more coat protein subunits are involved

in this process. Interestingly, we recently discovered

that packaging of the RNA genome is directly coupled

to replication of the genome, suggesting potential

approaches for packaging of foreign RNAs.

In other studies, we are investigating the mechanism

by which nodaviral protein B2 suppresses RNA

silencing in infected cells. Preliminary data indicate

that protein B2 binds to double-stranded RNA and that

it interferes with cleavage of double-stranded RNA

substrates by the cellular protein Dicer.

We are also collaborating with several investigators

at Scripps Research, the Salk Institute, and Harvard

University to develop nodaviruses as platforms for

delivery of anthrax antitoxins. To this end, we are

using particles to display the VWA domain of capillary

morphogenesis protein 2, the cellular receptor for anthrax

toxin, in a multivalent fashion on the surface of the

virion. Two insertion sites yielding different patterns of


180 copies of the VWA domain were selected on the

basis of computational modeling of the high-resolution

crystal structure of the insect nodavirus Flock House

virus. The resulting chimeric viruslike particles protect

cultured cells from the toxic effects of protective antigen

and lethal factor, 2 of the 3 proteins that make up

anthrax toxin. Experiments in animals are currently under

way to show that these particles also function as antitoxins

in vivo. This research is important because it illustrates

that protein domains containing more than 150

amino acids can be displayed on Flock House virus in

a biologically functional form, suggesting numerous additional

applications.

Flock House virus particles are also good candidates

for novel materials in nanotechnology applications. The

particles are stable, easily manipulated, biocompatible,

and nontoxic in vivo and can be produced easily and

in high quantities. The high-resolution x-ray structure

of the virus revealed the potential for using chemical

approaches to attach ligands to the surface of the virus

and for using genetic strategies to modify the capsid. In

collaboration with M. Manchester, Department of Cell

biology, and M. Ozkan, University of California, Riverside,

California, we used conjugation chemistry to couple

inorganic nanotubes and quantum dots to Flock

House virus particles to produce an array of novel hybrid

structures. This approach may one day be used to fabricate

unique materials for a variety of applications,

including biofilms with tunable pore sizes, 3-dimensional

scaffolds for production of nanoelectronic devices, and

drug delivery.

PUBLICATIONS

Portney, N.G., Singh, K., Chaudhary, S., Destito, G., Schneemann, A., Manchester, M.,

Ozka, M. Organic and inorganic nanoparticle hybrids. Langmuir 21:2098, 2005.

Schwarcz, W.D., Barroso, S.P., Gomes, S.M., Johnson, J.E., Schneemann, A.,

Oliveira, A.C., Silva, J.L. Virus stability and protein-nucleic acid interaction as

studied by high-pressure effects on nodaviruses. Cell. Mol. Biol. (Noisy-le-grand).

50:419, 2004.

Venter, P.A., Krishna, N.K., Schneemann, A. Capsid protein synthesis from replicating

RNA directs specific packaging of the genome of a multipartite, positivestrand

RNA virus. J. Virol. 79:6239, 2005.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Molecular Biology of

Retroviruses

J.H. Elder, A.P. de Parseval, Y.-C. Lin, S. de Rozieres,

U. Chatterji,* K. Tam, B.E. Torbett**

* Department of Immunology, Scripps Research

** Department of Molecular and Experimental Medicine, Scripps Research

Our research centers on the molecular characterization

of retroviruses, with emphasis on feline

immunodeficiency virus (FIV). FIV causes an

AIDS-like syndrome in domestic cats, and although it

does not infect humans, the feline retrovirus has many

structural and functional similarities to HIV, the causative

agent of AIDS in humans. Thus, study of FIV can

yield insights into ways to interfere with the retrovirus

life cycle that may ultimately result in the development

of treatments for infections in both cats and humans.

During the past year, we focused on 2 major areas:

the molecular characterization of cell-surface receptors

for FIV and the molecular basis for the development of

drug resistance in the aspartic protease encoded by FIV.

RECEPTOR STUDIES

MOLECULAR BIOLOGY 2005 221

Like many strains of HIV, FIV uses the chemokine

receptor CXCR4 to enter the primary target cell, the

CD4 + T cell. However, unlike HIV, FIV does not use

the cell-surface protein CD4 as a primary binding

receptor. Rather, the feline lentivirus initially binds to

another cell-surface molecule, CD134. In the past year,

we characterized the expression of CD134 and showed

that it is upregulated on CD4 + T cells. This observation

explains why FIV can infect and kill this subset of

T cells even though the virus’s surface glycoprotein does

not interact with CD4.

In an extension of these studies, we found that interaction

of the FIV surface glycoprotein gp95 with a soluble

version of CD134 allows the productive infection

of cells that bear the entry receptor, CXCR4, but lack

surface expression of the binding receptor, CD134. The

results are consistent with the notion that binding of

CD134 causes a conformational change in gp95, which

in turn increases the affinity of interaction with CXCR4

and facilitates infection of the target cell. These effects

are similar to the effects of binding of soluble CD4 by

gp120, the cell-surface glycoprotein of HIV, and indicate

that although different molecules are involved, the

actual mechanisms of infection of FIV and HIV are

strikingly similar. We speculate that the benefit of this

type of binding cascade is to limit exposure of critical


222 MOLECULAR BIOLOGY 2005

regions of the surface glycoproteins to the immune system

until the primary binding event has already occurred,

thus reducing the likelihood of virus neutralization.

We also precisely mapped regions of CD134 involved

in interaction with gp95. CD134 is a member of the

TNF-α receptor superfamily and has a domain structure

similar to that of the TNF-α receptor. Human CD134

does not bind FIV gp95, even though human CD134

shares considerable amino acid homology with feline

CD134. Using chimeric proteins consisting of feline

and human CD134 and site-directed mutagenesis, we

showed that as few as 3 amino acids in the C-terminal

part of outer domain 1 of feline CD134 are sufficient

to impart FIV gp95 binding and receptor function to

human CD134. Structural studies of both receptor and

ligand will establish a molecular basis for the putative

conformational change induced by interaction with the

binding receptor.

DEVELOPMENT OF DRUG RESISTANCE BY FIV

ASPARTIC PROTEASE

The aspartic protease of lentiviruses is a particularly

good target for drug therapy because its function in processing

the viral Gag and Pol polyproteins is absolutely

required for generation of infectious virus. Drugs active

against the HIV protease have been keys to the success

of highly active antiretroviral therapy used to treat

patients infected with HIV. The substrate and inhibitor

specificity of FIV differs from that of HIV, and we previously

reported the identification of amino acids that

define the different specificities. Comparing FIV with

HIV offers a means to better understand the development

of resistance to therapy, an ongoing problem with

current drugs used to treat HIV disease.

Interestingly, parallels exist between amino acid

positions that dictate differences in substrate specificity

between FIV and HIV aspartic protease and those

that mutate in response to drug treatment. Mutations

in these sites increase the dissociation constant for

complexes consisting of the protease and an inhibitor

drug, but at a cost in catalytic efficiency for the protease.

Over time, compensatory amino acid substitutions

occur that result in an increase in catalytic efficiency,

which results in increased expression of virus despite

drug treatment.

We prepared mutants of FIV protease in which amino

acids found in drug-resistant HIV protease were placed

in the equivalent positions in the FIV enzyme. These

“HIV-inized” FIV proteases had drug sensitivity profiles

similar to those of HIV protease. In studies with cells

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

transduced with gag/pol gene expression vectors encoding

HIV-FIV hybrid proteases, the Gag/Pol polyproteins

were processed with proper fidelity and had the expected

drug sensitivities. When engineered into FIV, these

hybrid proteases will offer a means to study drug resistance

and to develop new inhibitors capable of blocking

replication of drug-resistant viruses, without the

biohazard associated with handling infectious HIV.

PUBLICATIONS

de Parseval, A., Chatterji, U., Morris, G., Sun, P., Olson, A.J., Elder, J.H. Structural

mapping of CD134 residues critical for interaction with feline immunodeficiency

virus. Nat. Struct. Mol. Biol. 12:60, 2005.

de Parseval, A., Chatterji, U., Sun, P., Elder, J.H. Feline immunodeficiency virus

targets activated CD4 + T cells by using CD134 as a binding receptor. Proc. Natl.

Acad. Sci. U. S. A. 101:13044, 2004.

de Rozieres, S., Swan, C.H., Sheeter, D.A., Clingerman, K.J., Lin, Y.-C., Huitrón-

Reséndiz, S. Henriksen, S., Torbett, B.E., Elder, J.H. Assessment of FIV-C infection

of cats as a function of treatment with the protease inhibitor, TL-3.

Retrovirology 1:38, 2004.

Montes-Rodriguez, C.J., Alavez, S., Elder, J.H., Haro, R., Moran, J., Prospero-

Garcia, O. Prolonged waking reduces human immunodeficiency virus glycoprotein

120- or tumor necrosis factor α-induced apoptosis in the cerebral cortex of rats.

Neurosci. Lett. 360:133, 2004.

Metalloenzyme Engineering

D.B. Goodin, C.D. Stout, A.-M.A. Hays, S. Vetter,

E.C. Glazer, A.E. Pond, H.B. Gray,* J.R. Winkler,*

J.H. Dawson,** T.L. Poulos,*** M.A. Marletta****

* California Institute of Technology, Pasadena, California

** University of South Carolina, Columbia, South Carolina

*** University of California, Irvine, California

**** University of California, Berkeley, California

Our overall goals are to understand the fundamental

structural features of metalloenzyme catalysts

and to create catalysts for useful chemical reactions.

We use a number of techniques in structural biology

and spectroscopy and strategies of rational protein

redesign and molecular evolution. In the past year, we

made progress in several areas.

One area of recent interest has been the design

and use of molecular wires as probes for the active

sites of enzymes such as cytochrome P450 and nitric

oxide synthase (NOS). In an ongoing collaboration with

H.B. Gray, California Institute of Technology, we are

investigating the binding of these wires, which are

specifically designed substrate analogs linked to photochemical

or redox-active sensitizers, to the active

site of metalloproteins. The wires are being developed

to serve as reporters of the active-site environment and


as tools to allow rapid deposition or withdrawal of electrons

to drive redox catalysis. In addition, luminescent

wires that are quenched upon either binding or release

from the protein may be useful as imaging agents or as

tools for identifying novel enzyme inhibitors.

P450s make up a large family of enzymes responsible

for a vast range of biologically important oxidation

reactions in mammals, plants, fungi, and bacteria. An

important unresolved question concerns how the deeply

buried heme cofactor of these enzymes achieves regioselective

and stereoselective catalysis of a wide range

of substrates. In the past year, we completed a detailed

structural analysis by x-ray crystallography of cytochrome

P450 cam complexed with 2 sensitizer-linked

substrate probes, D4A and D8A. These probes differ

in length but bind identically at the substrate end of

the wire (Fig. 1).

Fig. 1. Crystal structure at 1.6 Å of P450cam containing D8A, a

synthetic molecular wire. The adamantyl substrate analog is observed

at the camphor binding site for wires of different lengths. Changes

in the F and G helices in response to wire length illustrate the conformational

flexibility in these regions that may be responsible for

the diversity of substrate recognition by P450s.

Significant changes in the protein structure near the

F and G helices accommodate the changes in linker

length. These changes are similar to those that may be

responsible for substrate-binding specificity of mammalian

P450s and indicate that prokaryotic enzymes

have similar conformational flexibility. These changes

also suggest the nature of the dynamic intermediates

that must exist transiently in solution during substrate

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 223

entry and product egress. The conformational change

associated with movement of the F and G helices is

transmitted to a backbone carbonyl at the active site

of the enzyme, which has been implicated in gating

the critical peroxy-bond cleavage that activates the

enzyme for catalysis.

In other studies, we are designing and synthesizing

specific pterin-based molecular wires for the active

site of NOS. NOSs are complex enzymes used for the

production of nitric oxide from arginine and play many

critical roles in biological signal transduction. As thiolate

coordinate heme enzymes, they have structural

and functional similarities to P450s. One unique feature

is the role played by the pterin cofactor of NOS.

Recent results suggest that the pterin donates an electron

to either the heme or the substrate at defined steps

in the catalytic mechanism. In the past year, we designed

and synthesized a series of pterin analogs tethered to

sensitizers containing redox-active ruthenium to be used

as specific molecular triggers and probes of the NOS

active site. In addition, we measured the FeIII/II and

FeII/I couples by direct cyclic voltammetry of inducible

NOS in organic films on graphite electrodes.

These studies allow easy and rapid measurements

of electron transfer between the enzyme and the electrode

surface and enabled us to detect the interconversion

of several coordination states of the enzyme.

These studies, coupled with the use of molecular wires

as mediators of electron transfer at electrode surfaces,

will provide a new way to prove the function of NOS

and related enzymes.

PUBLICATIONS

Hays, A.-M., Dunn, A.R., Chiu, R., Gray, H.B., Stout, C.D. Goodin, D.B. Conformational

states of cytochrome P450 cam revealed by trapping of synthetic molecular

wires. J. Mol. Biol. 344:455, 2004.

Udit, A.K., Belliston-Bittner, W., Glazer, E.C., Nguyen, Y.H.L., Gillon M., Hill,

M.G., Marletta, M.A., Goodin, D.B. Gray, H.B. Redox couples of inducible nitric

oxide synthase. J. Am. Chem. Soc. 127:11212, 2005.

Control of Cell Division

S.I. Reed, C. Baskerville, L.-C. Chuang, B. Grünenfelder,

M. Henze, J. Keck, V. Liberal, K. Luo, B. Olson,

S. Ekholm-Reed, S. Rudyak, O. Sangfelt, A. Smith,

C. Spruck, D. Tedesco, F. van Drogen, J. Wohlschlegel, V. Yu

Biological processes of great complexity can be

approached by beginning with a systematic

genetic analysis in which the relevant components

are first identified and the consequences of


224 MOLECULAR BIOLOGY 2005

selectively eliminating the components via mutations

are investigated. We use yeast, which is uniquely

tractable to this type of analysis, to investigate control

of cell division. In recent years, it has become apparent

that the most central cellular processes throughout

the eukaryotic phylogeny are highly conserved in terms

of both the regulatory mechanisms used and the proteins

involved. Thus, it has been possible in many instances to

generalize from yeast cells to human cells.

CONTROL IN YEAST

In recent years, we have focused on the role and

regulation of the Cdc28 protein kinase (Cdk1). Initially

identified by means of a mutational analysis of the yeast

cell cycle, this protein kinase and its analogs are ubiquitous

in eukaryotic cells and are central to a number

of aspects of control of cell-cycle progression.

One current area of interest is regulation of cellular

morphogenesis by Cdk1. The activity of Cdk1 driven

by mitotic cyclins modulates polarized growth in yeast

cells. Specifically, these activities depolarize growth by

altering the actin cytoskeleton. We found that several

proteins that modulate actin structure are targeted by

Cdk1, and we are investigating whether these phosphorylation

events control actin depolarization.

A second major area of interest is the regulation of

mitosis. A key aspect of mitotic regulation in yeast is

the accumulation of Cdc20, which triggers the transition

from metaphase to anaphase. Cdc20 is an essential

cofactor of the protein-ubiquitin ligase known as

the anaphase-promoting complex or APC/C. It is through

the ubiquitin-mediated proteolysis of a specific anaphase

inhibitor, securin (Pds1 in yeast), that anaphase

is initiated. We found that cells are prevented from entering

mitosis when DNA replication is blocked by the

drug hydroxyurea, which causes the destabilization of

Cdc20 and inhibition of Cdc20 translation.

While investigating mitosis, we found that Cks1, a

small Cdk1-associated protein, appears to regulate the

proteasome. Proteasomes are complex proteases that

target ubiquitylated proteins, including important cellcycle

regulatory proteins. Surprisingly, we found that

Cks1 regulates a nonproteolytic function of proteasomes,

the transcriptional activation of Cdc20. Specifically,

Cks1 is required to recruit proteasomes to the gene

CDC20 for efficient transcriptional elongation. Our investigations

of CDC20 led to the conclusion that Cks1 is

required for recruitment of proteasomes to and transcriptional

elongation of many other genes, as well.

Currently, we are elucidating the mechanism whereby

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Cks1 recruits proteasomes and facilitates transcriptional

elongation. Our most recent results suggest that Cks1

and proteasomes in conjunction with Cdk1 mediate

remodeling of chromatin.

CONTROL IN MAMMALIAN CELLS

We showed previously that the human homologs

of the Cdc28 protein kinase are so highly conserved,

structurally and functionally, relative to the yeast protein

kinase, that they can function and be regulated

properly in a yeast cell. Analyzing control of the cell

cycle in mammalian cells, we produced evidence for

the existence of regulatory schemes, similar to those

elucidated in yeast, that use networks of both positive

and negative regulators.

A principal research focus is the positive regulator

of Cdk2, cyclin E. Cyclin E is often overexpressed and/or

deregulated in human cancers. Using a tissue culture

model, we showed that deregulation of cyclin E confers

genomic instability, probably explaining the link to

carcinogenesis. The observation that deregulation of

cyclin E confers genomic instability led us to hypothesize

a mechanism of cyclin E–mediated carcinogenesis

based on accelerated loss of heterozygosity at tumor

suppressor loci. We are testing this hypothesis in transgenic

mouse models. We showed previously that a

cyclin E transgene expressed in mammary epithelium

markedly increases loss of heterozygosity at the p53

locus, leading to enhanced mammary carcinogenesis.

We are extending these investigations by using mouse

prostate, testis, and skin models.

In an attempt to understand cyclin E–mediated

genomic instability, we are investigating how deregulation

of cyclin E affects both S phase and mitosis. Recent

data suggest that deregulation of cyclin E impairs DNA

replication by interfering with assembly of the prereplication

complex. Cyclin E deregulation also impairs the

transition from metaphase to anaphase by promoting

the accumulation of mitotic checkpoint proteins.

Our interest in cyclin E deregulation in cancer led

us to examine the pathway for turnover of cyclin E.

We showed that phosphorylation-dependent proteolysis

of cyclin E depends on a protein-ubiquitin ligase

known as SCF hCdc4 . The F-box protein hCdc4 is the

specificity factor that targets phosphorylated cyclin E.

We are investigating how ubiquitylation of cyclin E is

coordinated with other processes required for its degradation.

We are also investigating SCF hCdc4 ubiquitylation

of other important cellular proteins.

Because of the functional relationship between

hCdc4 and cyclin E, we are studying the role of muta-


tions of hCDC4, the gene that encodes hCdc4, in carcinogenesis.

We found that hCDC4 is mutated and most

likely is a tumor suppressor in endometrial cancer and

breast cancer. In endometrial cancer, tumors with mutations

in hCDC4 are more aggressive than tumors without

mutations in this gene. Because we showed that

loss of hCdc4 leads to deregulation of cyclin E through

the cell cycle, these results confirm the observation that

in some cancers deregulation of cyclin E is associated

with aggressive disease and poor outcome.

Another area of interest is the role of Cks proteins

in mammals, complementing our research in yeast.

Mammals express 2 orthologs of yeast Cks1, known as

Cks1 and Cks2. Experiments in mice lacking the gene

for Cks1 and Cks2 revealed that each ortholog has a

specialized function. Cks1 is required as a cofactor for

Skp2-mediated ubiquitylation and turnover of inhibitors

p21, p27, and p130. Cks2 is required for the

transition from metaphase to anaphase in both male

and female meiosis I. Nevertheless, mice nullizygous

at the individual loci are viable. However, doubly nullizygous

mice have not been observed because embryos

die at the morula stage, a finding consistent with an

essential redundant function. We found that this function

most likely is involved in transcriptional elongation

and is linked to chromatin remodeling, as in yeast.

PUBLICATIONS

Huisman, S.M., Bales, O.A.M., Bertrand, M., Smeets, M.F.M.A., Reed, S.I.,

Segal, M. Differential contribution of Bud6p and Kar9p in microtubule capture and

spindle orientation in S. cerevisiae. J. Cell Biol. 167:231, 2004.

Reed, S.I. Cell cycle. In: Cancer: Principles and Practice of Oncology, 7th ed. DeVita

V.T., Jr., Hellman, S., Rosenberg, S.A. (Eds.). Lippincott Williams & Wilkins, Philadelphia,

2004, p. 83.

Reed, S.I., Rothman, J.H. Cell division, growth and death [editorial]. Curr. Opin.

Cell Biol. 16:599, 2004.

Spruck C.H., Smith, A.P.L., Ekholm-Reed, S., Sangfelt, O., Keck, J., Strohmaier, H.,

Méndez, J., Widschwendter, M., Stillman, B., Zetterberg A., Reed, S.I. Deregulation

of cyclin E and genomic Instability. In: Hormonal Carcinogenesis IV. Li, J.J., Li,

S.A., Llombart-Bosch, A. (Eds.). Springer, New York, 2004, p. 98.

Wittenberg, C., Reed, S.I. Cell cycle-dependent transcription in yeast: promoters,

transcription factors, and transcriptomes. Oncogene 24:2746, 2005.

Wohlschlegel, J.A., Johnson, E.S., Reed, S.I., Yates, J.R. III. Global analysis of protein

sumoylation in Saccharomyces cerevisiae. J. Biol. Chem. 279:45662, 2004.

Yu, V.P.C.C., Baskerville, C., Grünenfelder, B., Reed, S.I. A kinase-independent

function of Cks1 and Cdk1 in regulation of transcription. Mol. Cell 17:145, 2005.

Yu, V.P.C.C., Reed, S.I. Cks1 is dispensable for survival in Saccharomyces cerevisiae.

Cell Cycle 3:1402, 2004.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Regulating Cell Proliferation:

Flipping Transcriptional and

Proteolytic Switches

C. Wittenberg, M. Ashe, R. de Bruin, M. Guaderrama,

B.-K. Han, T. Kalashnikova, N. Spielewoy

MOLECULAR BIOLOGY 2005 225

Cell proliferation is governed primarily by controlling

the activities of positive and negative

regulators of cell-cycle transitions. Inhibitors

of cyclin-dependent protein kinase (CDK) and the positive

regulatory subunits, cyclins, are critical in establishing

the proper timing of cell-cycle transitions and in

imposing cell-cycle checkpoints. The activities of those

proteins are largely regulated via periodic transcriptional

activation coupled with regulated proteolysis. We

focus primarily on those regulatory mechanisms.

As in animal cells, initiation of the cell cycle in the

budding yeast Saccharomyces cerevisiae occurs during

late G 1 phase and is governed by the controlled accumulation

of G 1 CDK activity. A large family of G 1 -specific

genes, including those for the G 1 cyclins Cln1 and

Cln2, are coordinately regulated by 2 transcription factors:

SBF and MBF. As in metazoans, the transcriptional activation

of those genes depends on the activity of a distinct

G 1 cyclin, Cln3, that acts on promoter-bound

transcription factors to promote recruitment of components

of the RNA polymerase II complex.

By analogy with metazoan Rb, an inhibitor of the

E2F transcription factor that is antagonized by cyclin

D/CDK, we predicted the existence of a G 1 -specfic

transcriptional repressor that is inactivated by Cln3/CDK.

Using the combined application of molecular genetics

and mass spectrometry–based multidimensional protein

identification technology, we identified an SBF-specific

transcriptional repressor, Whi5, that is inactivated via

phosphorylation by Cln3/CDK. This discovery provides

a unifying mechanism for initiation of the cell cycle in

yeast and metazoans.

We also identified several other transcriptional regulators,

including Nrm1, a novel cell cycle–dependent

repressor of MBF-dependent transcription. Rather than

repressing expression early in the cell cycle as Whi5

does, Nrm1 acts as cells pass into S phase, thereby limiting

MBF-dependent gene expression to the G 1 phase.

Because expression of the gene for NRM1 depends on

MBF, the gene cannot act until MBF becomes active.

Consequently, the gene confers negative autoregulation


226 MOLECULAR BIOLOGY 2005

on MBF. Additional factors associated with the 2 transcription

factors are under investigation.

One of the primary roles of G 1 cyclin-associated

CDKs is to promote the ubiquitin-dependent proteolysis

of cell-cycle regulators, including the G 1 cyclins

themselves. CDK-dependent phosphorylation of a number

of proteins targets the proteins for recognition by the

Cdc34-SCF ubiquitin ligase complex. Grr1, one of several

distinct F box proteins that associate with that complex,

confers recognition of specific phosphorylated

targets. We are interested in the molecular basis of

that recognition. Previously, we showed that the interaction

between Grr1 and Cln2 requires basic residues

residing in the pocket of the leucine-rich repeat of Grr1

and defined a transferable “degron” in the C terminus

of Cln2 that is phosphorylated by the CDK. These findings,

combined with our understanding of the mechanisms

that govern G 1 -specific transcription, indicate that

an integrated autoregulatory circuit governs the events

of G 1 phase and ensures the orderly progression of

events in the cell cycle.

In addition to its role in cell-cycle control, SCF Grr1

plays a central role in regulating the expression of genes

induced by glucose and amino acids. We showed that

the glucose signal promotes ubiquitin-mediated proteolysis

of Mth1, which is required for maintenance of

transcriptional repression of glucose-inducible genes.

Glucose triggers phosphorylation of Mth1 by casein

kinase I, thereby promoting recognition by SCF Grr1 .

Surprisingly, recognition of phosphorylated Mth1 requires

properties of Grr1 distinct from those required for recognition

of phosphorylated G 1 cyclins. The same properties

are also important for Grr1-dependent recognition

of an as yet unknown target required for the activation

of amino acid–regulated genes via SPS signaling. Efforts

are under way to identify novel targets of Grr1 and to

investigate the possibility that Grr1 mediates the coordination

of cell-cycle progression with the availability

of environmental nutrients.

PUBLICATIONS

Flick, K., Wittenberg, C. Multiple pathways for suppression of mutants affecting

G 1 -specific transcription in Saccharomyces cerevisiae. Genetics 169:37, 2005.

Wittenberg, C. Cell cycle: cyclin guides the way. Nature 434:34, 2005.

Wittenberg, C., Reed, S.I. Cell cycle-dependent transcription in yeast: promoters,

transcription factors, and transcriptomes. Oncogene 24:2746, 2004.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Cell-Cycle Checkpoints,

DNA Repair, and Oxidative

Stress Response

P. Russell, C. Chahwan, S. Coulon, L.-L. Du, P.-H. Gaillard,

V. Martin, T. Nakamura, C. Noguchi, E. Noguchi,

M. Rodriguez, P. Shanahan, K. Tanaka, H. Zhao

The cellular responses to DNA damage and cytotoxic

stress are highly conserved through evolution.

A fortunate consequence of this conservation is

that “simple” eukaryotes such as the fission yeast

Schizosaccharomyces pombe can be used as model

systems for more complex multicellular organisms. We

use S pombe to study cell-cycle checkpoints, DNA

repair, and stress response mechanisms. Defects in

these mechanisms underlie a number of human diseases,

including cancer.

DNA REPLICATION CHECKPOINT

The challenging task of replicating a eukaryotic

genome is often made more difficult by conditions that

interfere with progression of the replisome, the complex

formed by the close association of the key proteins used

during DNA replication. Protein complexes bound to

DNA, chemical adducts in DNA, and deoxyribonucleotide

starvation are among the situations that can impede

replisomes. The DNA replication checkpoint senses

stalled replication forks and directs cellular responses

that help preserve the integrity of the genome. One

of these responses is the S-M checkpoint. This checkpoint

delays the onset of mitosis (M phase) while DNA

synthesis (S phase) is under way, thereby providing time

to recover from stalled forks. The same checkpoint also

controls how damaged DNA is replicated.

DNA-dependent protein kinases, such as ATM and

ATR in humans and Rad3 in fission yeast, are central

components of the replication checkpoint. Acting in

conjunction with regulatory subunits (e.g., Rad26 in

fission yeast) and other protein complexes, these kinases

activate checkpoint effector kinases. The effector of

the replication checkpoint in fission yeast is Cds1

(Chk2). A few years ago, we discovered mediator of

replication checkpoint-1 (Mrc1), an adaptor or mediator

protein that directs the replication checkpoint signal

from Rad3 to Cds1. We recently discovered that

the forkhead-associated domain of Cds1 mediates the

binding of Cds1 to Mrc1. This interaction allows Rad3

to activate Cds1.


Cds1 controls repair systems that are required to

tolerate stalled replication forks. We hope to better

understand these systems by identifying proteins that

associate with the forkhead-associated domain of Cds1.

Mus81, a novel protein related to the XPF nucleotide

excision repair protein, was identified in a screen for

such proteins. We found that Mus81 associates with

another protein, Eme1, to form a structure-specific

endonuclease that resolves X-shaped Holliday junctions.

In recent studies with T. Wang, Stanford University,

Stanford, California, and M.N. Boddy, Department of

Molecular Biology, we discovered that phosphorylation

of Mus81 by Cds1 helps preserve genome integrity

when replication forks arrest. We hypothesize that the

phosphorylation prevents the Mus81-Eme1 complex

from cleaving stalled replication forks.

Stalled forks are potentially unstable structures

prone to rearrangement and collapse. We previously

reported that the protein Swi1 helps preserve stalled

forks and is necessary for strong activation of Cds1.

Recent studies with J.R. Yates, Department of Cell

Biology, indicated that Swi1 associates with Swi3 to

form a fork-protection complex. We found that the

complex travels with the replisome during DNA replication.

It is therefore ideally placed to detect, stabilize,

and signal stalled replication forks (Fig. 1). We

speculate that Swi1 and Swi3 homologs in humans

have equivalent functions.

Fig. 1. Stabilization of stalled replication forks. The fork-protection

complex (FPC), which consists of Swi1 and Swi3, travels with

the replisome. Mrc1 also appears to travel with the fork. When the

replisome stalls at obstructions in the fork or for other reasons, the

fork-protection complex and Mrc1 are required for activation of Cds1

by Rad3-Rad26 kinase. The Rad9-Rad1-Hus1 (9-1-1) complex is

also required for Cds1 activation.

DNA DAMAGE CHECKPOINT

The DNA damage checkpoint prevents the onset of

mitosis when DNA is damaged (Fig. 2). This checkpoint

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

is enforced by the protein kinase Chk1, which is activated

by Rad3. Activation of Chk1 requires the adaptor protein

Crb2. Crb2 is rapidly recruited to double-stranded

breaks in DNA. We recently found that Rad3 and Tel1

(the ATM homolog in fission yeast) stimulate Crb2

recruitment by phosphorylating histone H2A at the

DNA break site. We also found that the tandem C-terminal

BRCT domains in Crb2 are essential for Crb2

homo-oligomerization.

Recently, we investigated how Tel1/ATM is recruited

for sites of DNA damage and how it is activated. These

studies, done in collaboration with T. Hunter, the Salk

Institute, La Jolla, California, revealed that Tel1/ATM

interacts with the extreme C terminus of Nbs1. Nbs1

is a subunit of the Mre11-Rad50-Nbs1 complex that

associates with and processes double-stranded breaks.

We found that the interaction with Nbs1 is essential

for ATM activation.

OXIDATIVE STRESS RESPONSE

MOLECULAR BIOLOGY 2005 227

Fig. 2. The unicellular yeast S pombe divides by medial fission

(top left panel). It has 3 chromosomes and approximately 4000

genes. The DNA damage checkpoint arrests division in cells

exposed to ionizing radiation (+IR) (top right panel). Pulse-field gel

electrophoresis shows that the chromosomes are fragmented by

120 Gy of ionizing radiation (bottom panel). About 3 hours are

required to repair the DNA, necessitating a checkpoint that prevents

mitosis while DNA repair is under way.

Oxidative stress caused by reactive oxygen species

can be highly toxic, causing damage to proteins, lipids,


228 MOLECULAR BIOLOGY 2005

and nucleic acids. Oxidative stress elicits a complex

gene expression response that is orchestrated in large

part by MAP kinase cascades. The fission yeast Spc1

MAP kinase pathway is homologous to the p38 pathway

in humans. We recently discovered Csx1, a protein

that collaborates with Spc1 to control gene expression

in response to oxidative stress. Csx1 is an RNA-binding

protein that mediates global control of gene expression

in response to oxidative stress by binding and stabilizing

mRNA that encodes Atf1, a transcription factor that

is also regulated by Spc1. Most recently, we focused

on a newly discovered family of proteins that interact

with Csx1.

PUBLICATIONS

Du, L.L., Moser, B.A., Russell, P. Homo-oligomerization is the essential function of

the tandem BRCT domains in the checkpoint protein Crb2. J. Biol. Chem.

279:38409, 2004.

Kai, M., Boddy, M.N., Russell, P., Wang, T.S.F. Replication checkpoint kinase

Cds1 regulates Mus81 to preserve genome integrity during replication stress.

Genes Dev. 19:919, 2005.

McGowan, C.H., Russell, P. The DNA damage response: sensing and signaling.

Curr. Opin. Cell Biol. 16:629, 2004.

Nakamura, T.M., Moser, B.A., Du, L.L., Russell, P. Cooperative control of Crb2 by

ATM-family and Cdc2 kinases is essential for the DNA damage checkpoint in fission

yeast. Mol. Cell. Biol., in press.

Noguchi, E., Noguchi, C., McDonald, W.H., Yates, J.R. III, Russell, P. Swi1 and

Swi3 are components of a replication fork protection complex in fission yeast. Mol.

Cell. Biol. 24:8342, 2004.

Tanaka, K., Russell, P. Cds1 phosphorylation by Rad3-Rad26 kinase is mediated by

forkhead-associated domain interaction with Mrc1. J. Biol. Chem. 279:32079, 2004.

You, Z., Chahwan, C., Bailis, J., Hunter, T. Russell, P. ATM activation and its recruitment

to damaged DNA require binding to the C terminus of Nbs1. Mol. Cell. Biol.

25:5363, 2005.

Zhao, H., Russell, P. DNA binding domain in the replication checkpoint protein

Mrc1 of Schizosaccharomyces pombe. J. Biol. Chem. 279:53023, 2004.

DNA Damage Responses in

Human Cells

C.H. McGowan, V. Blais, H. Gao, E. Langley, A. MacLaren,

J. Scorah, E. Taylor

Complex multicellular organisms, such as humans,

have large numbers of mitotically competent cells

that are capable of renewal, repair, and, to some

extent, regeneration. The advantages of being able to

replace damaged or aged cells are off set by the inherent

susceptibility of mitotic cells to acquiring mutations and

becoming cancerous. DNA is inherently vulnerable to

many sorts of chemical and physical modification; thus,

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

as they duplicate and divide, cells can acquire mutations.

Both spontaneous and induced DNA damage must

be repaired with minimal changes if growth, renewal,

and repair are to be successful. Our overall objective is

to understand how mammalian cells protect themselves

from DNA damage and thus from cancer.

Eukaryotic cells have evolved with a complex network

of DNA repair processes and cell-cycle checkpoint

responses that ensure that damaged DNA is repaired

before it is replicated and becomes fixed in the genome.

These pathways are highly conserved through evolution,

and much information about human responses to DNA

damage has been gained from studies of simple genetically

tractable organisms such as yeast. We use a combination

of molecular, cellular, and genetic techniques

to determine how these pathways operate in human cells.

Checkpoints control the order and timing of events

in the cell cycle; they ensure that biochemically independent

processes are coupled so that a delay in a critical

cell-cycle process will cause a delay in all other aspects

of progression of the cycle. In addition, checkpoints

coordinate repair with delays in progression of the cell

cycle and promote the use of the most appropriate repair

pathway. We used genetic models to identify 2 checkpoint

kinases in humans that limit progression of the

cell cycle when DNA is damaged. One of these kinases,

Chk2, is activated in response to DNA damage. Chk2

physically interacts with Mus81-Eme1, a conserved

DNA repair protein that has homology to the xeroderma

pigmentosum F family of endonucleases. Xeroderma

pigmentosum is a cancer-prone disorder that results

from a failure to appropriately repair damaged DNA.

Biochemical analysis indicates that Mus81-Eme1

has associated endonuclease activity against structurespecific

DNA substrates, including Holliday junctions.

Enzymatic analysis, immunofluorescence studies, and the

use of RNA interference have all contributed to the conclusion

that Mus81-Eme1 is required for recombination

repair in human cells. We are also using gene targeting to

study the function of the Mus81-Eme1 endonuclease in

mice. Inactivation of Mus81 in mice increases genomic

instability and sensitivity to DNA damage but does not

promote tumorogenesis. In addition, we showed that

Mus81-Eme1 is specifically required for survival after

exposure to cisplatin, mitomycin C, and other commonly

used anticancer drugs. As a point of interaction between

checkpoint control and DNA repair, the relationship

between Mus81-Eme1 and Chk2 most likely will provide

information critical to understanding the responses to

DNA damage as a whole.


Anticancer therapy is largely based on the use of

genotoxic agents that damage DNA and thus kill dividing

cells. Coordination of cell-cycle checkpoints and

DNA repair is especially important when unusually

high amounts of DNA damage occur after radiation or

genotoxic chemotherapy. Hence, a detailed understanding

of cellular responses to DNA damage is essential

to understanding both the development and the treatment

of disease in humans.

PUBLICATIONS

Dendouga, N., Gao, H., Moechars, D., Janicot, M., Vialard, J., McGowan, C.H.

Disruption of murine Mus81 increases genomic instability and DNA damage sensitivity

but does not promote tumorigenesis. Mol. Cell. Biol. 25:7569, 2005.

McGowan, C.H., Russell, P. The DNA damage response: sensing and signaling.

Curr Opin. Cell Biol. 16:629, 2004.

Zhang, R., Sengupta, S., Yang, Q,, Linke, S.P., Yanaihara, N., Bradsher, J., Blais, V,.

McGowan, C.H., Harris, C.C. BLM helicase facilitates Mus81 endonuclease activity

in human cells. Cancer Res. 65:2526, 2005.

DNA Repair and the Maintenance

of Genomic Stability

M.N. Boddy, Y. Pavlova, S. Pebenard, G. Raffa

DNA repair pathways have evolved to protect the

genome from ever-present genotoxic agents.

Highlighting the importance of the pathways,

defects in DNA repair mechanisms strongly predispose

the host to cancer and to neurologic and developmental

disorders. The DNA repair systems we study in fission

yeast are evolutionarily conserved, and therefore

our investigations provide a valuable framework for

understanding genome maintenance in human cells.

Although many DNA repair mechanisms have been

described, information on how they are coordinated

with necessary changes in chromatin structure is limited.

We are studying the essential structural maintenance

of chromosomes (SMC) complex Smc5-Smc6.

The molecular functions of Smc5-Smc6 are unknown,

but the complex is related to the SMC complexes that

hold replicated sister chromatids together (cohesin)

and condense chromatin before its segregation at

mitosis (condensin).

In collaboration with J.R. Yates, Department of Cell

Biology, we purified the Smc5-Smc6 complex and determined

the identity of the core components. The holocomplex

consists of the Smc5-Smc6 heterodimer and

6 additional non-SMC elements, Nse1–Nse6 (Fig. 1).

We expressed and purified individual components of

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 229

Fig. 1. Architecture of the Smc5-Smc6 holocomplex. Nse1, Nse3,

and Nse4 form a stable heterotrimer that then associates with

Smc5. Nse2 interacts directly with Smc5 in the absence of the

other Nse proteins. Smc6 interacts directly with Smc5 but none of

the other components. Nse5 and Nse6 form a stable heterodimer

that also binds directly to Smc5. Double-headed arrows indicate

interactions between subcomplexes. Nse5-Nse6 may recruit the

holocomplex to stalled replication forks and certain DNA damage

sites (black oval on leading-strand template of replication fork).

the complex and determined the architecture of the

holocomplex. Nse1–Nse4 are essential for growth, and

hypomorphic mutants of these proteins cause cellular

sensitivity to genotoxic agents such as ultraviolet light

and x-rays. Nse5 and Nse6 are nonessential, but cells

lacking either protein also are hypersensitive to DNAdamaging

agents. Notably, Nse1 and Nse2 contain

certain zinc finger domains that implicate these 2 elements

in the modification of target proteins with ubiquitin

and the small ubiquitin-like protein SUMO. Such

protein modifications play roles in DNA repair and

chromatin remodeling.

Our genetic analyses support a role for the Smc5-

Smc6 complex in stabilizing replication forks that have

stalled at sites of DNA damage. We have also identified

a critical role for the Smc5-Smc6 complex in meiosis,

the process that generates gametes for reproduction

and genetic diversity. A critical feature of meiosis is

the programmed formation of DNA double-strand breaks

followed by repair of the breaks via homologous recombination.

We found that the Smc5-Smc6 complex functions

in the correct repair of the breaks and that mutants


230 MOLECULAR BIOLOGY 2005

of Smc5-Smc6 do not segregate homologous chromosomes

at the first meiotic division.

Finally, we identified a physical interaction between

the Smc5-Smc6 complex and Rad60, an essential DNA

repair factor required for the homologous recombination

repair of DNA. Rad60 is regulated by the replication

checkpoint, and thus we can study the important

but poorly defined interface between DNA repair and

cell-cycle checkpoints.

PUBLICATIONS

Kai, M., Boddy, M.N., Russell, P., Wang, T.S. Replication checkpoint kinase Cds1

regulates Mus81 to preserve genome integrity during replication stress. Genes Dev.

19:919, 2005.

Pebernard, S., McDonald, W.H., Pavlova, Y., Yates, J.R., III, Boddy, M.N. Nse1,

Nse2, and a novel subunit of the Smc5-Smc6 complex, Nse3, play a crucial role in

meiosis. Mol. Biol. Cell 15:4866, 2004.

Delineation of Oncogenic and

Tumor-Suppressing Pathways

via Genetic Approaches

P. Sun, Q. Deng, C. Kannemeier, R. Liao, B. Moser

Our major interests are the genetic alterations

involved in tumorigenesis and the cellular pathways

that must be altered during oncogenic

transformation. To this end, we analyzed the behaviors

of primary, normal human cells after stable transduction

of oncogenes, such as ras and MPM2.

Members of the ras family of oncogenes encode

small GTP-binding proteins that transduce growth signals.

Constitutive activation of ras often occurs in tumors

and contributes to tumor development. In normal cells,

activation of ras triggers an antioncogenic response

called premature senescence, a stable growth arrest

that must be overcome before transformation occurs.

We showed that ras induces senescence through sequential

activation of 2 MAP kinase pathways. Initially, ras

activates the MAP kinase kinase (MEK)–extracellular signal–regulated

kinase (ERK) pathway. Sustained activation

of MEK-ERK turns on the stress-induced p38 pathway,

which subsequently causes senescence.

These results revealed a novel, tumor-suppressing

function of p38, in addition to its known roles in inflammation

and stress responses. In other studies, we identified

additional signaling components, either upstream or

downstream of p38, that mediate premature senescence.

To determine how premature senescence is bypassed

in tumors, we dissected the functions of an adenovi-

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

rus-encoded oncoprotein, E1A, that can rescue cells

from ras-induced senescence. E1A directly binds to

and inhibits the functions of several cellular proteins,

such as members of the Rb family, p300/CBP, and p400,

that have been implicated in tumor-suppressing pathways.

Our results indicated that senescence-bypassing

activity resides in the N terminus of E1A and requires

binding of both Rb and p300/CBP, but not binding of

p400. Although interference with the p16 INK4A /Rb

pathway or with p300/CBP functions alone did not

result in bypassing of senescence, these 2 types of

genetic alterations complemented mutants of E1A with

defects in Rb binding and p300/CBP binding, respectively,

to rescue cells from ras-induced senescence and

lead to cellular transformation. Therefore, genetic alterations

that disrupt the p16 INK4A /Rb pathway and those

that perturb the p300/CBP functions cooperate to

bypass ras-induced senescence. These results indicate

that p300 and CBP are integral components of the

senescence pathway. Both p300 and CBP have tumorsuppressing

functions. The critical role of p300 and

CBP in the senescence response has provided a mechanistic

basis for the tumor-suppressing function of

these proteins.

Another focus of our research is MDM2, an oncogene

that can mediate transformation primarily through inactivation

of the tumor suppressor protein p53. However, we

found that MDM2 confers resistance to a growth-inhibitory

cytokine, transforming growth factor β, through a

p53-independent mechanism. Currently, we are delineating

this p53-independent activity of MDM2, which

may play an important role in tumorigenesis.

In other studies, we are systematically searching

for genetic alterations that contribute to specific tumorassociated

phenotypes, such as drug resistance, cellular

immortalization, and metastasis. For these investigations,

we are using cDNA expression libraries or libraries

of short interfering RNAs.

PUBLICATIONS

de Parseval, A., Chatterji, U., Morris, G., Sun, P., Elder, J.H. Fine mapping of

CD134 residues critical for interaction with feline immunodeficiency virus. Nat.

Struct. Mol. Biol. 12:60, 2005.

de Parseval, A., Chatterji, U., Sun, P., Elder, J.H. Feline immunodeficiency virus

targets activated CD4 + T cells by using CD134 as a binding receptor. Proc. Natl.

Acad. Sci. U. S. A. 101:13044, 2004.

de Parseval, A., Ngo, S., Sun, P., Elder, J.H. Factors that increase the effective

concentration of CXCR4 dictate feline immunodeficiency virus tropism and kinetics

of replication. J. Virol. 78:9132, 2004.

Deng, Q., Li, Y., Tedesco, D., Liao, R., Fuhrmann, G., Sun, P. The ability of E1A to

rescue ras-induced premature senescence and confer transformation relies on inactivation

of both p300/CBP and Rb family proteins. Cancer Res. 65:8298, 2005.


The 5-HT 7 Receptor as a Target

in Depression and Schizophrenia

P.B. Hedlund, P.E. Danielson, S. Huitrón-Reséndiz,

S.J. Hendriksen, S. Semenova, M.A. Geyer, A. Markou,

J.G. Sutcliffe

Serotonin (5-HT) is produced by a small group of

nuclei in the brain stem that send their projections

to a vast number of receptive fields. The family

of receptors for 5-HT is the most diverse family that

binds a single ligand; it has at least 14 members. One

of these is the 5-HT 7 receptor, which we previously discovered.

In earlier studies, we showed that this receptor

mediates resetting of circadian rhythms by the hypothalamus.

Despite vast differences in amino acid sequence

between the 5-HT 7 receptor and the 5-HT 1A receptor,

the 2 share considerable pharmacology and have been

implicated in some of the same functions. 5-HT 1A is

more abundant than 5-HT 7 , but the areas of the brain

that express the 2 receptors overlap considerably.

We produced mutant mice in which the gene for

the 5-HT 7 receptor was inactivated. Studies with these

mice and SB-266970, a 5-HT 7 -selective antagonist,

indicated that this receptor mediates serotonin-induced

hypothermia and is important for fine tuning of temperature

homeostasis.

Sleep, circadian rhythm, and mood are related phenomena.

5-HT 7 -selective antagonists increase REM sleep

latency and decrease the cumulative duration of REM

sleep, patterns the opposite of those found in patients

with clinical depression. Several antidepressants activate

5-HT 7 neurons in the circadian control area of the

hypothalamus, and chronic treatment with antidepressants

diminishes both activation and 5-HT 7 binding

there. We examined sleep parameters in the mutant

mice in which the gene for the 5-HT 7 receptor was

inactivated. We found that they spent less time than

normal mice in REM sleep. This pattern is the opposite

of that found in humans with depression.

Two models of behavioral despair, the forced swim

test and the tail suspension test, make rats and mice

immobile. This immobility, or helplessness, is likened

to depression in humans because a high correlation

exists between the ability of antidepressant drugs to

reverse immobility in rodents and to be effective clinically

in humans. Furthermore, mice selectively bred to

have increased helplessness in these behavioral despair

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 231

tests resemble patients with clinical depression. The

mice have decreased REM latency and more cumulative

REM sleep, elevated levels of corticosterone, a decreased

5-HT metabolism index, and altered serotonin-induced

hypothermia. We examined unmedicated 5-HT 7 mutant

mice in these tests and found that the mice remained

significantly more mobile than unmedicated normal

mice during both the forced swim and the tail suspension

tests. Normal mice medicated with the 5-HT 7 -selective

antagonist SB-266970 mimicked the mobility of

unmedicated mutant mice, whereas the selective antagonist

had no effect on the mobility of mutant mice. A

selective serotonin reuptake inhibitor increased mobility

in both types of mice (albeit at a lower concentration

in the mutant mice), suggesting that the inhibitor

worked through an independent mechanism.

These results are consistent with the notion that

the 5-HT 7 mutant mice have characteristics of a partially

“antidepressed” state: they spend less time in

REM sleep, have reduced immobility in the forced swim

and tail suspension tests, and have decreases in serotonin-induced

hypothermia. Normal mice medicated

with 5-HT 7 -selective antagonists resemble unmedicated

5-HT 7 mutant mice in these measures. These findings

suggest that 5-HT 7 -selective antagonists might be sufficient

treatment for some aspects of clinical depression.

Several antipsychotic drugs have high affinity for

the 5-HT 7 receptor. We examined the role of 5-HT 7

receptors in an animal model of schizophrenia: phencyclidine-induced

disruption of prepulse inhibition of

the acoustic startle reflex. In untreated mice, we

found no difference between mice in which the gene

for the 5-HT 7 receptor was inactivated and wild-type

mice in startle response or in prepulse inhibition regardless

of prepulse intensity, interstimulus interval, or pulse

intensity. SB-269970 had no effect on prepulse inhibition.

The disruption of prepulse inhibition produced

by phencyclidine in wild-type mice did not occur in

the mutant mice. Similarly, the effect of phencyclidine

on prepulse inhibition was reduced by SB-269970 in

wild-type mice. The results indicate a specific role for

the 5-HT 7 receptor in the glutamatergic prepulse inhibition

model of schizophrenia.

PUBLICATIONS

de Lecea, L., Sutcliffe, J.G. Hypocretin as a wakefulness regulatory peptide. In:

The Orexin/Hypocretin System: Physiology and Pathophysiology. Nishino, S., Sakurai,

T. (Eds.). Humana Press, Totowa, NJ, 2005, p. 143. A volume in the series

Contemporary Clinical Neuroscience.

de Lecea, L., Sutcliffe, J.G. (Eds.). The Hypocretins: Integrators of Physiological

Functions. Plenum Press, New York, 2005.


232 MOLECULAR BIOLOGY 2005

Hedlund, P.B., Huitrón-Reséndiz, S., Henriksen, S.J., Sutcliffe, J.G. 5-HT 7 receptor

inhibition and inactivation induce antidepressantlike behavior and sleep pattern.

Biol. Psychiatry, in press.

Hedlund, P.B., Sutcliffe, J.G. Functional, molecular and pharmacological advances

in 5-HT 7 receptor research. Trends Pharmacol. Sci. 25:481, 2004.

Hedlund, P.B., Sutcliffe, J.G. 5-HT 7 receptors as favorable pharmacological targets

for drug discovery. In: The Serotonin Receptors: From Molecular Pharmacology to

Human Therapeutics. Roth, B.L. (Ed.). Humana Press, Totowa, NJ, in press.

Sutcliffe, J.G., de Lecea, L. The hypocretin/orexin system. In: Handbook of Contemporary

Neuropharmacology. Sibley, D. (Ed.). Wiley & Sons, Hoboken, NJ, in press.

Sutcliffe, J.G., de Lecea, L. Hypocretins/orexins in brain function. In: Handbook of Neurochemistry

and Molecular Neurobiology. Lim, R. (Ed.). Springer, New York, in press.

Sutcliffe J.G., de Lecea, L. Not asleep, not quite awake. Nat. Med. 10:673, 2004.

Ziolkowska, B., Gieryk, A., Bilecki, W., Wawrzczak-Bargiela, A., Wedzony, K.,

Chocyk, A., Danielson, P.E., Thomas, E.A., Hilbush, B.S., Sutcliffe, J.G.,

Przewlocki, R. Regulation of α-synuclein expression in limbic and motor brain

regions of morphine-treated mice. J. Neurosci. 25:4996, 2005.

Molecular Neurobiology of

CNS Disorders

E.A. Thomas, J.G. Sutcliffe, P.A. Desplats, S. Narayan,

K.E. Kass, W. Huang

GENE EXPRESSION IN STRIATAL DISORDERS

We have identified and cataloged approximately

50 genes that are predominantly expressed

in the striatum in the brain. Our long-standing

hypothesis is that such genes most likely encode

proteins that are preferentially associated with particular

physiologic processes in the striatum and therefore

may be relevant to striatal disorders. Using oligonucleotide

microarrays, we measured expression of these

genes simultaneously in the striatum of R6/1 mice, a

transgenic model of Huntington’s disease.

A total of 81% of striatal genes had increased

expression in mice in presymptomatic and/or symptomatic

stages of illness. Changes in expression of genes

associated with G protein signaling and calcium homeostasis

are of particular interest for future studies. The

most striking decrease occurred in β4, a newly identified

subunit of the sodium channel. Changes in expression

began when the mice were 8 weeks old, and expression

had progressively decreased almost 10-fold by the time

the mice were 8 months old. Two novel sequences with

highly specific striatal expression also had differences in

expression throughout the life span of the mutant mice,

as determined by in situ hybridization analysis.

Expression differences of 15 of the striatum-enriched

genes were tested in rats treated with 6-hydroxydopamine,

a rodent model of Parkinson’s disease. No

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

changes in expression were detected in any of the

genes tested.

These findings indicate that mutant huntingtin

protein causes selective deficits in the expression of

mRNAs responsible for striatum-specific physiologic

changes. Furthermore, the results suggest that although

both Huntington’s disease and Parkinson’s disease

involve striatal dysfunction, the differences in the

molecular pathologic changes associated with the 2

diseases are distinct.

MOLECULAR MARKERS OF SCHIZOPHRENIA

Schizophrenia is a life-long mental illness with variable

expression and unknown etiology. The major clinical

manifestations of schizophrenia at the time of onset

of the illness are psychotic symptoms; however, as the

illness progresses, the negative symptoms become more

predominant. In addition, many other neurologic aspects

change during the course of the illness. We are interested

in the molecular factors that influence manifestation

of the symptoms and the course of schizophrenia

after its onset and how treatment modifies the effects

of illness.

Using oligonucleotide microarrays, we generated

gene expression profiles from tissue samples obtained

at autopsy from the prefrontal cortex of patients with

schizophrenia of short and long duration. Because correct

treatment early in the illness is thought to have a

beneficial effect on the outcome of schizophrenia, the

identification of genes involved in the early and late

stages of disease will be important for understanding

the progression of the illness.

PUBLICATIONS

Dean, B., Keriakous, D., Thomas, E.A., Scarr, E. Understanding the pathology of

schizophrenia: the impact of high-throughput screening of the genome and proteome

in postmortem CNS. Curr. Psychiatry Rev. 1:1, 2005.

Digney, A., Keriakous, D., Scarr, E., Thomas, E.A., Dean, B. Differential changes

in apolipoprotein E in schizophrenia and bipolar I disorder. Biol. Psychiatry

57:711, 2005.

Yao, J.K., Thomas, E.A., Reddy, R.D., Keshavan, M.S. Association of plasma

apolipoproteins D with RBC membrane arachidonic acid levels in schizophrenia.

Schizophr. Res. 72:259, 2005.

Ziolkowska, B., Gieryk, A., Bilecki, W., Wawrzczak-Bargiela, A., Wedzony, K.,

Chocyk, A., Danielson, P.E., Thomas, E.A., Hilbush, B.S., Sutcliffe, J.G.,

Przewlocki, R. Regulation of α-synuclein expression in limbic and motor brain

regions of morphine-treated mice. J. Neurosci. 25:4996, 2005.


Molecular Biology of Sleep

L. de Lecea, C. Suzuki, C. Pañeda, B. Boutrel,* R. Winsky-

Sommerer, A. Coda, S. Huitrón-Reséndiz,* A.J. Roberts,*

J.G. Sutcliffe, G.F. Koob,* S.J. Henriksen*

* Molecular and Integrative Neurosciences Department, Scripps Research

Our goal is to understand the cellular and molecular

components that modulate cortical activity

and sleep. In particular, we focus on the characterization

of neuropeptides first described by our group:

cortistatin and the hypocretins.

CORTISTATIN

Cortistatin is a neuropeptide expressed in the cerebral

cortex. Of its 14 residues, 11 also occur in the

neuropeptide somatostatin. However, cortistatin and

somatostatin have different physiologic functions. Cortistatin

is neuroinhibitory and promotes sleep.

We generated mice deficient in cortistatin and determined

their behavioral profile in collaboration with

A.J. Roberts, Molecular and Integrative Neurosciences

Department. Because cortistatin has anticonvulsant

activity, we tested seizure susceptibility in cortistatindeficient

mice. We also did gene array studies to determine

the consequences of cortistatin deficiency in mice

lacking the gene for this neuropeptide. Our results

suggest that cortistatin has multiple functions in the

maintenance of cortical excitability.

THE HYPOCRETINS

The hypocretins, 2 neuropeptides derived from the

same precursor, are produced in a few thousand cells

in the lateral part of the hypothalamus. The hypocretins

are key molecules for the stability of the states of vigilance.

Lack of hypocretin peptides or hypocretin-producing

neurons produces narcolepsy, a sleep disorder

characterized by uninvited intrusions of sleep into wakefulness.

Patients with narcolepsy experience excessive

daytime sleepiness and cataplexy, a sudden loss of

muscle tone upon certain stimuli. Recent studies

indicated that patients with narcolepsy lack hypocretin-expressing

cells, suggesting that narcolepsy is a

neurodegenerative disease of the hypocretinergic system.

In anatomic and electrophysiologic experiments,

we found that neurons expressing hypocretin are contacted

by neurons expressing corticotropin-releasing

factor (CRF), a major component of the stress response.

Hypocretin neurons contain CRF receptors. Intracellular

recordings in hypothalamic slices from transgenic

mice that express green fluorescent protein under the

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 233

control of the hypocretin promoter indicated that CRF

depolarizes hypocretin neurons through the CRF 1 receptor.

Further, hypocretin neurons are not activated upon

stress in mice that lack the gene for this receptor. These

data suggest a close association between the CRF and

hypocretin systems in the acute stress response.

Because CRF is involved in addiction and because

hypocretin neurons project to key areas involved in brain

reward, we hypothesized that hypocretin neurons might

be involved in addiction-related behaviors. We found

that hypocretin-1 leads to the reinstatement of previously

extinguished cocaine-seeking behavior but does

not alter cocaine intake in rats. In collaboration with

P.J. Kenny and A. Markou, Molecular and Integrative

Neurosciences Department, we discovered that hypocretin-1

negatively regulates the activity of brain reward

circuitries. Hypocretin-induced reinstatement of cocaine

seeking can be prevented by simultaneous blockade of

noradrenergic and CRF systems but not by blockade of

either system alone. These findings reveal a previously

unidentified role for hypocretins in drug craving and

relapse behavior. Moreover, hypocretins may drive drug

seeking through induction of a negative affective state

by activation of stress pathways in the brain.

NEUROPEPTIDE S

Neuropeptide S is a newly discovered neuropeptide

expressed prominently in a few hundred neurons in the

area near the locus coeruleus. We found that infusion of

neuropeptide S into the brain ventricles in mice dramatically

enhanced wakefulness and suppressed anxiety. The

neuropeptide activated several brain nuclei related to

arousal. We showed that neurons expressing neuropeptide

S project to and depolarize neurons expressing hypocretin.

Our data strongly suggest that neuropeptide S

is an important modulator of sleep and waking.

PUBLICATIONS

de Lecea, L. Reverse genetics and the study of sleep. In: Sleep: Circuits and Functions.

Luppi, P.-H. (Ed.). CRC Press, Boca Raton, FL, 2004, p. 109.

de Lecea, L., Sutcliffe, J.G. The hypocretins and sleep. FEBS J., in press.

de Lecea, L., Sutcliffe, J.G. (Eds.) Hypocretins: Integrators of Physiological Functions.

Springer, New York, 2005.

Huitrón-Reséndiz, S., Kristensen, M.P., Sánchez-Alavez, M., Clark, S.D., Grupke,

S.L., Tyler, C., Suzuki, C., Nothacker, H.P., Civelli, O., Criado, J.R., Henriksen, S.J.,

Leonard, C.S., de Lecea, L. Urotensin II modulates rapid eye movement sleep

through activation of brainstem cholinergic neurons. J. Neurosci. 25:5465, 2005.

Levine, A.S., Winsky-Sommerer, R., Huitrón-Reséndiz, S., Grace, M.K., de Lecea, L.

Injection of neuropeptide W into paraventricular nucleus of hypothalamus increases

food intake. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288:R1727, 2005.

Martin, G., Guadaño-Ferraz, A., Morte, B., Ahmed, S., Koob, G.F., de Lecea, L.

Siggins, G.R. Chronic morphine treatment alters N-methyl-D-aspartate receptors in

freshly isolated neurons from nucleus accumbens. J. Pharmacol. Exp. Ther.

311:265-73, 2004.


234 MOLECULAR BIOLOGY 2005

Pañeda, C., Winsky-Sommerer, R., Boutrel, B., de Lecea, L. The corticotropinreleasing

factor-hypocretin connection: implications in stress response and addiction.

Drug News Perspect. 18:250, 2005.

Spier, A.D., Fabre, V., de Lecea, L. Cortistatin radioligand binding in wild-type and

somatostatin receptor-deficient mouse brain. Regul. Pept. 124:179, 2005.

Sutcliffe, J.G., de Lecea L. Not asleep, not quite awake. Nat. Med. 10:673, 2004.

Tallent, M.K., Fabre, V., Qiu, C., Calbet, M., Lamp, T., Baratta, M.V., Suzuki, C.,

Siggins, G.R., Henriksen, S.J., Criado, J.R., Roberts, A., de Lecea, L., Cortistatin

overexpression in transgenic mice produces deficits in synaptic plasticity and learning.

Mol. Cell. Neurosci., in press.

Ureña, J.M., La Torre, A., Martínez, A., Lowenstein, E., Franco, N., Winsky-Sommerer,

R., Fontana, X., Casaroli-Marano, R., Ibáñez-Sabio, M.A., Pascual, M., del

Rio, J.A., de Lecea, L., Soriano, E. Expression, synaptic localization, and developmental

regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in

the developing and adult brain. J. Comp. Neurol. 490:119, 2005.

Winsky-Sommerer, R., Boutrel, B., de Lecea , L. Stress and arousal: the corticotropin-releasing

factor/hypocretin circuitry. J. Mol. Neurobiol., in press.

Winsky-Sommerer, R., Yamanaka, A., Diano, S., Borok, E., Roberts, A., Sakurai, T.,

Kilduff, T.S., Horvath, T.L., de Lecea, L. Interaction between the corticotropinreleasing

factor system and hypocretins (orexins): a novel circuit mediating stress

response. J. Neurosci. 24:11439, 2004.

Xu, Y., Reinscheid, R.R., Huitrón-Reséndiz, S., Clark, S.D., Wang, Z., Lin, S.H.,

Brucher, F.A., Zeng, J., Ly, H.K., Henriksen, S.J., de Lecea, L., Civelli, O. Neuropeptide

S: a novel neuropeptide promoting arousal and anxiolytic-like effects.

Neuron 43:487, 2004.

Molecular Neuroscience:

Lysophospholipid Signaling,

Neural Aneuploidy

J. Chun, B. Almeida, B. Anliker, E. Birgbauer, M. Fontanoz,

S. Gardell, C. Paczkowski, D. Herr, D. Kaushal, G. Kennedy,

M. Kingsbury, C.W. Lee, M. McConnell, M. McCreight,

S. Peterson, S. Rehen, R. Rivera, M. Lu, W. Westra,

A.H. Yang, X.Q. Ye, Y. Yung, L. Zhu

Understanding the nervous system—how it arises

developmentally and how it carries out its myriad

complex tasks in normal and diseased states—

is a major challenge. We are studying 2 topics with

both basic and potentially therapeutic relevance: the

role of lysophospholipid signaling and the role of genomic

alterations within individual neurons as manifested

by aneuploidy.

LYSOPHOSPHOLIPID SIGNALING

Lysophospholipids are simple phospholipids containing

a glycerophosphate or glycerosphingoid backbone

and single acyl chain of varied length and saturation.

Two major forms of lysophospholipids are lysophosphatidic

acid and sphingosine 1-phosphate (Fig. 1). It

is now clear from our research and that of many oth-

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

Fig. 1. Chemical structures of lysophosphatidic acid and sphingosine

1-phosphate.

ers that most important actions of lysophospholipids

are mediated by cognate G protein–coupled receptors.

A growing range of neurobiological functions is being

identified, particularly effects on Schwann cells and oligodendrocytes,

which are involved in myelination, and

on neuroprogenitor cells of the cerebral cortex. To

determine receptor selectivity and actual neurobiological

function, we are producing mice that lack the genes

for single and multiple receptors. In collaboration with

other scientists at Scripps Research, we are developing

chemical tools to dissect the in vivo function of lysophosphatidic

acid and sphingosine 1-phosphate.

During 2004, the range of new biological functions

for receptor-mediated lysophospholipid signaling continued

to grow. With collaborators from around the world,

we showed that lysophospholipid signaling influences

the cardiovascular system, the immune system, cancer

cell motility, and, especially, neuropathic pain and

multiple sclerosis.

Neuropathic pain is pain due to nerve damage or

dysfunction. Mechanisms for the initiation of this type

of pain are poorly understood. In a murine model of

neuropathic pain, activation of a single lysophospholipid

receptor was necessary for the initiation of pain;

such pain did not develop in mice that lacked the gene

for the receptor. Another medically important disease,

multiple sclerosis, can be approximated in animals by

immunization with myelin antigens to produce experimental

autoimmune encephalomyelitis. Agonists for

lysophospholipid receptors (specifically, sphingosine

1-phosphate receptor agonists) abrogated the disability

normally produced by experimental autoimmune

encephalomyelitis, suggesting a role for this signaling

pathway in the medical biology and a possible therapy


for multiple sclerosis. We are expanding these themes

in previously identified and new biological systems.

NORMAL NEURAL ANEUPLOIDY

Are all neurons of the brain genetically identical,

as is widely assumed, or are differences encoded within

individual genomes? Using a combination of spectral

karyotyping, which “paints” chromosomes to allow their

unambiguous detection, and fluorescence in situ hybridization,

which uses labeled point-probes to identify discrete

genetic loci in interphase cells, we detected a

substantial degree of genomic variation in the normal

brain. During neurogenesis, approximately one third of

all cells are aneuploid, produced, at least in part, by

chromosome missegregation mechanisms. In postmitotic

neurons, in which spectral karyotyping cannot be

used because neurons are in interphase, fluorescence

in situ hybridization of sex chromosomes revealed a

high percentage of aneuploidy, and the total number

of aneuploid cells is certainly higher if the remaining

autosomes are considered (Fig. 2).

Fig. 2. Examples of neural aneuploidy in different regions of the

brain in adult mice as revealed by fluorescence in situ hybridization.

During 2004, by analyzing mice deficient in DNA

surveillance or repair molecules, we detected a new

influence on the generation of aneuploidy. One of these

molecules, the mutated protein ATM, is the cause of

the rare genetic disease ataxia-telangiectasia. Elimination

of the gene for ATM or the gene for XRCC5, another molecule

involved in DNA surveillance and repair, resulted

in major increases in the number and severity of aneuploid

neural progenitor/stem cells, indicating a positive

biological link between aneuploidy and molecules involved

with genome integrity. Currently, we are exploring the

basic phenomenologic aspects and functional importance

of neural aneuploidy during development and in

disease processes.

PUBLICATIONS

Anliker, B., Chun, J. Cell surface receptors in lysophospholipid signaling. Semin.

Cell Dev. Biol. 15:457, 2004.

Anliker, B., Chun, J. Lysophospholipid G protein-coupled receptors. J. Biol. Chem.

279:20555, 2004.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 235

Baudhuin, L.M., Jiang, Y., Zaslavsky, A., Ishii, I., Chun, J., Xu, Y. S1P 3 -mediated

Akt activation and cross-talk with platelet-derived growth factor receptor (PDGFR).

FASEB J. 18:341, 2004.

Chun, J. Choices, choices, choices. Nat. Neurosci. 7:323, 2004.

Girkontaite, I., Sakk, V., Wagner, M., Borggrefe, T., Tedford, K., Chun, J., Fischer,

K.-D. The sphingosine-1-phosphate (S1P) lysophospholipid receptor S1P 3 regulates

MAdCAM-1 + endothelial cells in splenic marginal sinus organization. J. Exp.

Med. 200:1491, 2004.

Hama, K., Aoki, J., Fukaya, M., Kishi, Y., Sakai, T., Suzuki, R., Ohta, H., Yamori, T.,

Watanabe, M., Chun, J., Arai, H. Lysophosphatidic acid and autotaxin stimulate

cell motility of neoplastic and non-neoplastic cells through LPA1. J. Biol. Chem.

279:17634, 2004.

Inoue, M., Rashid, M.H., Fujita, R., Contos, J.J., Chun, J., Ueda, H. Initiation of

neuropathic pain requires lysophosphatidic acid receptor signaling [published correction

appears in Nat. Med. 10:755, 2004]. Nat. Med. 10:712, 2004.

Ishii, I., Fukushima, N., Ye, X., Chun, J. Lysophospholipid receptors: signaling and

biology. Annu. Rev. Biochem. 73:321, 2004.

Kingsbury, M.A., Rehen, S.K., Ye, X., Chun, J. Genetics and cell biology of lysophosphatidic

acid receptor-mediated signaling during cortical neurogenesis. J. Cell. Biochem.

92:1004, 2004.

Levkau, B., Hermann, S., Theilmeier, G., van der Giet, M., Chun, J., Schober, O.,

Schäfers, M. High-density lipoprotein stimulates myocardial perfusion in vivo. Circulation

110:3355, 2004.

McConnell, M.J., Kaushal, D., Yang, A.H., Kingsbury, M.A., Rehen, S.K., Treuner, K.,

Helton, R., Annas, E.G., Chun, J., Barlow, C. Failed clearance of aneuploid embryonic

neural progenitor cells leads to excess aneuploidy in ATM-deficient but not the

Trp53-deficient adult cerebral cortex. J. Neurosci. 24:8090, 2004.

Nofer, J.-R., van der Giet, M., Tölle, M., Wolinska, I., von Wnuck-Lipinski, K.,

Baba, H.A., Gödecke, A., Tietge, U.J., Ishii, I., Kleuser, B., Schäfers, M., Fobker, M.,

Zidek, W., Assmann, G., Chun, J., Levkau, B. HDL induces NO-dependent vasorelaxation

via the lysophospholipid receptor S1P 3 . J. Clin. Invest. 113:569, 2004.

Rao, T.S., Lariosa-Willingham, K.D., Lin, F.-F. Yu, N., Tham, C.-S., Chun, J., Webb, M.

Growth factor pre-treatment differentially regulates phosphoinositide turnover downstream

of lysophospholipid receptor and metabotropic glutamate receptors in cultured

rat cerebrocortical astrocytes. Int. J. Dev. Neurosci. 22:131, 2004.

Sanna, M.G., Liao, J., Jo, E., Alfonso, C., Ahn, M.Y., Peterson, M.S., Webb, B.,

Lefebvre, S., Chun, J., Gray, N., Rosen, H. Sphingosine 1-phosphate (S1P) receptor

subtypes S1P 1 and S1P 3 , respectively, regulate lymphocyte recirculation and

heart rate. J. Biol. Chem. 279:13839, 2004.

Webb, M., Tham, C.-S., Lin, F.-F., Lariosa-Willingham, K., Yu, N., Hale, J., Mandala,

S., Chun, J., Rao, T.S. Sphingosine 1-phosphate receptor agonists attenuate

relapsing-remitting experimental autoimmune encephalitis in SJL mice. J. Neuroimmunol.

153:108, 2004.

Chemical Glycobiology in the

Immune System

J.C. Paulson, P. Bengtson, O. Blixt, B.E. Collins, S. Han,

T. Islam, H. Tateno, Q. Yan

We investigate the roles of glycan-binding proteins

that mediate cellular processes central

to immunoregulation and human disease. We

work at the interface of biology and chemistry to understand

how the interaction of glycan-binding proteins

with their ligands modulates the functions of the pro-


236 MOLECULAR BIOLOGY 2005

teins in cell-cell adhesion and cell signaling. Projects

fall into 2 main areas: (1) functions of glycan-binding

proteins expressed on leukocytes and (2) regulation of

the synthesis of the carbohydrate ligands of the proteins

during leukocyte activation and differentiation. Our multidisciplinary

approach is complemented by a diverse

group of chemists, biochemists, cell biologists, and

molecular biologists.

SIGLEC FAMILY OF CELL ADHESION PROTEINS

A total of 11 human and 8 mouse siglecs have been

identified so far, and most siglecs are expressed on

leukocytes. The siglecs are a subfamily of the immunoglobulin

superfamily. They have variable numbers of

extracellular Ig domains, including a unique, homologous

N-terminal Ig domain that confers the ability to bind

to sialic acid–containing carbohydrate groups (sialosides)

of glycoproteins and glycolipids. The cytoplasmic

domains of the siglecs typically contain one or more

immunoreceptor tyrosine-based inhibitory motifs characteristic

of accessory proteins that regulate transmembrane

signaling of cell-surface receptor proteins.

To dissect the biology of the siglecs, we use novel

carbohydrate probes that modulate the function of the

proteins. We use chemoenzymatic approaches to synthesize

sialoside analogs recognized by siglecs. The

analogs range from potent inhibitors to multivalent

probes of siglec binding to monovalent sialic acid analogs

that can be fed to cells and incorporated into cell-surface

glycoproteins to add chemical functionality or alter the

affinity of sialoside ligands for cell-surface siglecs. Projects

on several members of the siglec family are ongoing.

CD22 (siglec-2) is an accessory molecule of the

B-cell receptor complex; it has both positive and negative

effects on receptor signaling. The carbohydrate

ligand recognized by CD22 is the sequence sialic acid

α-2-6-galactose, which commonly terminates N-linked

carbohydrate groups of glycoproteins. Significantly, ablation

of the gene that encodes β-galactoside α-2,6-sialyltransferase

I, the enzyme responsible for synthesis of

this carbohydrate in mice, causes a marked deficiency

in antibody production in response to vaccination with

T cell–dependent or T cell–independent antigens, establishing

the importance of the ligand in CD22 function.

We developed a novel method for in situ photoaffinity

cross-linking of CD22 to its ligands on the same cell

(cis) or on an adjacent cell (trans); we use a 9-arylazide-sialic

acid that is taken up by cells and incorporated

into cell-surface glycoproteins, allowing the

glycoproteins to be cross-linked to CD22 upon expo-

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

sure to ultraviolet light (Fig. 1). The striking finding is

that microdomain localization of CD22, not glycan

structure alone, strongly influences the glycoprotein

ligands CD22 interacts with, providing insights into

how glycan ligands influence the function of this molecule.

This basic observation on siglec-ligand interactions

most likely is recapitulated by other members of

the siglec family.

Fig. 1. Bioengineering of cell-surface glycoproteins to carry 9-arylazide-sialic

acids for in situ photoaffinity cross-linking of CD22 to

its ligands.

Other members of the siglec family differ from CD22

both in cellular distribution and in specificity for recognition

of sialic acid–containing oligosaccharides. We are

evaluating the roles of siglec-7 and siglec-9 in regulation

of human T-cell receptor signaling, the role of siglec-F

in the biology of eosinophils in mice, and the role of

myelin-associated glycoprotein (siglec-4) in regulation

of neurite formation. We recently developed a novel

approach in which a robotically printed glycan array is

used for combinatorial assessment of the effects of sialoside

analogs on the affinity of siglecs. This method

should be a rapid one for developing high-affinity sialoside

probes for each of the siglecs to facilitate investigations

into siglec biology.

REGULATION OF LEUKOCYTE GLYCOSYLATION

Activation of lymphocytes and other leukocytes

induces programmed changes in glycosylation. Such

changes regulate leukocyte trafficking and can modulate

the functions of carbohydrate-binding proteins. We

are systematically investigating the changes in glycosylation

that occur in B and T lymphocytes after activation

in order to elucidate the underlying molecular

mechanisms for these changes and their biological relevance.

To this end, in collaboration with S. Head and

the Consortium for Functional Glycomics (http://www

.functionalglycomics.org), we participated in the development

and use of a custom microarray of glycosyl-


transferase genes, and in collaboration with A. Dell,

Imperial College London, London, England, we correlated

dramatic changes in gene expression with changes

in the glycan profiles of the resting and activated B

and T cells.

PUBLICATIONS

Amado, M., Yan, Q., Comelli, E.M., Collins, B.D. Paulson, J.C. Peanut agglutinin

high phenotype of activated CD8 + T cells results from de novo synthesis of CD45

glycans. J. Biol. Chem. 279:36689, 2004.

Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M.E., Alvarez, R., Bryan,

M.C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D.J., Skehel, J.J.,

van Die, I., Burton, D.R., Wilson, I.A., Cummings, R., Bovin, N., Wong, C-H.,

Paulson, J.C. Printed covalent glycan array for ligand profiling of diverse glycan

binding proteins. Proc. Natl. Acad. Sci. U. S. A. 101:17033, 2004.

Blixt, O., Vasiliu, D., Allin, K., Jacobsen, N., Warnock, D., Razi, N., Paulson, J.C.,

Bernatchez, S., Gilbert, M., Wakarchuk, W. Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides

GD3, GT3, GM2, GD2, GT2, GM1, and GD1a.

Carbohydr. Res. 340:1963, 2005.

Bryan, M.C., Fazio, F., Lee, H.-K., Huang, C.-Y., Chang, A., Best, M.D., Calarese, D.A.,

Blixt, O., Paulson, J.C., Burton, D., Wilson, I.A., Wong, C.-H. Covalent display of

oligosaccharide arrays in microtiter plates. J. Am. Chem. Soc. 126:8640, 2004.

Collins, B.E., Paulson, J.C. Cell surface biology mediated by low affinity multivalent

protein-glycan interactions. Curr. Opin. Chem. Biol. 8:617, 2004.

Goldberg, D., Sutton-Smith, M., Paulson, J.C., Dell, A. Automatic annotation of

matrix-assisted laser desorption/ionization N-glycan spectra. Proteomics 5:865, 2005.

Han, S., Collins, B.E., Bengtson, P., Paulson, J.C. Homomultimeric complexes of CD22

in B cells revealed by protein-glycan cross-linking. Nat. Chem. Biol. 1:93, 2005.

Ikehara, Y., Ikehara, S.K., Paulson, J.C. Negative regulation of T cell receptor signaling

by Siglec-7 (p70/AIRM) and Siglec-9. J. Biol. Chem. 279:43117, 2004.

Kitazume, S., Nakagawa, K., Oka, R., Tachida, Y., Ogawa, K., Luo, Y., Citron, M.,

Shitara, H., Taya, C., Yonekawa, H., Paulson, J.C., Miyoshi, E., Taniguchi, N.,

Hashimoto, Y. In vivo cleavage of α2,6-sialyltransferase by Alzheimer’s β-secretase.

J. Biol. Chem. 280:8589, 2005.

Vyas, A.A., Blixt, O., Paulson, J.C., Schnaar, R.L. Potent glycan inhibitors of

myelin-associated glycoprotein enhance axon outgrowth in vitro. J. Biol. Chem.

280:16305, 2005.

Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

MOLECULAR BIOLOGY 2005 237


Published by TSRI Press ®. ©Copyright 2005,

The Scripps Research Institute. All rights reserved.

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