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
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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.