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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- Published by TSRI Press ®. ©Copyright 2005, 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
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Structure, Function, and Applicatio
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An Icosahedral Scaffold for Biophys
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The 5-HT 7 Receptor as a Target in
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Molecular Biology of Sleep L. de Le
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