News and Publications

Structural Biology of Protein-Ligand Interactions

I.A. Wilson, E.A. Stura, R.L. Stanfield, K.C. Garcia, T.A. Cross, W.L. Densley, M. Degano, S.E. Greasley, K. Gruber, M.R. Haynes, A. Heine, T. Horton, K. Hotta, M. Huang, O. Livnah, N. Mattsson, E.E. Ollmann, K.A. Renner, V.M. Reyes, C.E.A. Scott, B.W. Segelke, M. Skidmore, J.A. Speir, R.S. Stefanko, Y. Su, D.B. Williams, C. Wingren, X. Wu, J. Xu, M.M. Yamashita, K. Zeng

Our laboratory is interested in interactions and signal transduction events between proteins and their ligands. Current projects include x-ray crystallographic studies of antibody-antigen complexes, complexes of T-cell receptors (TCRs) with MHC molecules and peptide antigens, classical and nonclassical MHC molecules and their antigens, human growth factors and their receptors, and enzyme targets for chemotherapy in complex with small molecule inhibitors. A major focus is research on enzyme catalysis and signal transduction. These structure-based studies incorporate the design of small molecules as agonists or antagonists of receptors or as inhibitors of key regulatory enzymes.

CATALYTIC ANTIBODIES

The crystal structure of 13G5, an antibody to metallocene that catalyzes the disfavored exo Diels-Alder reaction, has been determined at 1.95-Å resolution for the unbound form and for the antibody in complex with a ferrocene derivative. Docking experiments with transition-state analogs and substrates suggest why the exo pathway is preferred to the endo pathway.

The structure of 33F12, an aldolase antibody, in complex with a transition-state analog has been determined at 2.05-Å resolution. The hydrophobic environment of the binding pocket may account for the unusual pK_a (~6.0) and for the reactivity of an essential lysine in the active site (Fig. 1). Three related aldolase antibodies all contain the same conserved lysine. X-ray data for a related natural aldolase enzyme have also been collected to 1.8-Å resolution.

Antibody 5C8 catalyzes the strongly disfavored 6-endo-tet cyclization of a trans-epoxy-alcohol, thereby violating Baldwin's rules for ring-closure reactions. We determined the structures to 2.5- and 2.0-Å resolution, respectively, of the unbound Fab and of the Fab bound to 2 inhibitors. Results of studies with an ab initio calculated model of the transition state of the reaction were consistent with general acid-base catalysis involving AspH95 and HisL89. Sequence comparisons with 3 antibodies that have the same activity and 4 antibodies that strongly bind the transition-state analog but are catalytically inactive corroborated these findings.

Antibody 21D8 is a catalytic antibody designed to investigate the role in enzymatic catalysis of the medium effect in which a change in solvent can dramatically alter the reaction rate. The structures of the unbound form of the antibody and of the antibody bound to its hapten were determined at 2.5- and 1.6-Å resolution, respectively. Further studies to explain the observed substrate specificity and high rate acceleration are in progress.

Antibody 1E9 catalyzes an endo Diels-Alder reaction between N-ethylmaleimide and tetrachlorothiophene dioxide with the highest catalytic efficiency of all reported catalytic antibodies. The structure of a 1E9-hapten complex has been determined at 1.9-Å resolution. Analysis of the structure and structural comparisons of 1E9 with other antibodies within the same family suggest that the high catalytic efficiency is due to the proximity effect, in which the substrates are brought together and bound in their proper orientation.

The catalytic antibody projects are being done in collaboration with R. Lerner of the Departments of Chemistry and Molecular Biology; K. Janda, D. Hilvert, and C.-H. Wong of the Department of Chemistry; and C. Barbas III and C. Shevlin of the Department of Molecular Biology.

ANTIBODIES TO HIV TYPE 1

We are studying a group of antipeptide antibodies that can neutralize several isolates of HIV type 1. These antibodies recognize the principal neutralizing determinant, or V3 loop, of the viral surface glycoprotein gp120. Three different neutralizing antibodies (50.1, 59.1, and 58.2) determined previously in complex with peptide antigens led to a model for the tertiary structure of the V3 loop. Data were also collected for a peptide complex with Fab 83.1, a broadly neutralizing antibody that recognizes several different viral isolates. We are using surface plasmon resonance to investigate the kinetic properties of antibodies 50.1, 58.2, 59.1, and 83.1. The goal of this project is to determine whether conformational variation of the V3 loop can be linked to use of chemokine receptors and hence to cell tropism.

MHC MOLECULES

The structural basis for differences in the stability of peptide binding and in T-cell recognition between the wild-type murine class I MHC molecule H-2K^b and 2 of its mutants is being investigated. High-resolution data have been collected at the Stanford Synchrotron Radiation Laboratory, Stanford, California, for K^bm1 and K^bm8 mutants in complex with viral peptides. These new structures, at 1.8- to 2.0-Å resolution, are expected to yield information about the subtle but important structural changes between mutant and wild-type H-2K^b that lead to major biological outcomes. This work is being done in collaboration with L. Teyton, Department of Immunology, and A. Brünmark and P. Peterson, R.W. Johnson Pharmaceutical Research Institute.

In an effort to understand the antigenic and structural properties of posttranslationally modified peptides presented to the cellular immune system, we determined the x-ray structure of H-2K^b with a synthetic glycopeptide (RGY-6h-Gal₂) at 2.1-Å resolution in collaboration with M. Jondal of the Karolinska Institute in Sweden (Fig. 2). The carbohydrate extends beyond the binding groove and is highly exposed for recognition by the TCR. For MHC presentation of peptide-linked haptens to the T-cell population, the linker length (i.e., exposure of hapten) is a critical factor in dictating the type of immune response. This information can be used to design glycosylated peptides suitable for initiating immune responses to unique carbohydrate antigens present in microbial infections and in transformed cells.

In collaboration with J. Stevens, E. Joly, and G. Butcher of the Babraham Institute in England, we crystallized rat class I MHC RT1.A^a in complex with a 13-residue mitochondrial peptide. This complex represents the first antigen target for cytotoxic T lymphocytes defined in this species at the molecular level and is involved in several causes of histoincompatibility. Complexes with different peptides are also being studied. Together, these structures will indicate any species-related differences between rat and human or mouse MHCs and reveal differences in binding between longer peptides and the shorter peptides (8--9 amino acids) studied so far.

Class II MHC molecules play a central role in the immune response by presenting peptide antigens for surveillance by T cells. A soluble form of the murine class II MHC molecule I-A^d was produced with a leucine zipper tail added to each chain to enhance dimer assembly and secretion in S2 cells. We determined the structure of I-A^d covalently linked to an ovalbumin peptide and to an influenza virus hemagglutinin peptide at 2.6- and 2.4-Å resolution, respectively. The floor of the peptide-binding groove contains an unusual ß-bulge (Fig. 3). Unlike the situation in other MHC-peptide complexes, the peptides do not insert any large anchor residues into the binding pockets of the shallow I-A^d peptide-binding groove.

Nonclassical class I MHC molecules have evolved for specific tasks that differ from those of classical class I MHC molecules, including the binding of nonpeptide antigens and peptides that differ from peptides that bind to classical MHC molecules. The nonclassical MHC class I molecules T10 and T22 are the ligands for G8, a T-cell clone. Heterodimers consisting of (1) T10 and ß₂-microglobulin and (2) T22 and ß₂-microglobulin are thought to be recognized by and stimulatory to G8 without presenting any peptide or nonpeptide moieties. T10 and T22 may thus represent a way for MHC-like molecules to adopt a peptide-free structure but still function with a different role in the immune system. X-ray data have been collected for T10 and T22 in collaboration with Y. Chien, Stanford University.

CD1 is another nonclassical MHC-like molecule that is encoded outside the MHC. The human CD1b isoform, part of the group I CD1 molecules, can present hydrophobic antigens such as lipids and lipoglycans to a subset of T lymphocytes. Therefore, CD1 molecules can play a crucial role in the outcome of bacterial infections. The group II CD1 molecules, including the human CD1d and murine CD1.1 isoforms, can also bind hydrophobic antigens, including peptides, and restrict the response of a subset of T lymphocytes with a distinct cytokine secretion pattern. We determined the structure of the murine CD1.1 molecule to 2.8-Å resolution. CD1.1 resembles the class I MHC molecules but has a larger, hydrophobic groove that could accommodate hydrophobic ligands. Although the protein was crystallized in the absence of added antigen, the groove is occupied with a ligand that appears to be lipid. We are attempting to cocrystallize mouse CD1.1 with a specific glycolipid to obtain structural information on the requirements for restrictions of lipid antigens presented to T cells. We are also pursuing the structure determination of 2 other human CD1 isoforms, CD1b and CD1d. These studies are being done in collaboration with R. Modlin, University of California, Los Angeles; M. Kronenburg, La Jolla Institute of Allergy and Immunology; and M. Brenner and S. Porcelli, Harvard Medical School.

T-CELL RECEPTOR

The TCR is a heterodimeric glycoprotein expressed on the surface of T lymphocytes. The central event in immune responses mediated by T cells is TCR recognition of peptide antigens from foreign pathogens in the context of the MHC. The interactions between TCRs and MHC molecules are finely modulated, because discriminating between self and foreign antigens is important. In collaboration with Dr. Teyton, we determined the structure of the first murine TCR (2C) to 2.5-Å resolution and of its complex with K^b/dEV8, a syngeneic MHC molecule with a self-peptide, to 3.2-Å resolution. We are actively pursuing structural studies of other TCR-peptide-MHC complexes to understand the molecular details of this complex system.

GUANINE NUCLEOTIDE DISSOCIATION INHIBITOR

Rab proteins play a central role in vesicular trafficking and are members of the Ras superfamily. They have an on state when bound to GTP and an off state when GTP is hydrolyzed into GDP. Although this molecular switch is regulated via many peripheral molecules, guanine nucleotide dissociation inhibitor is the most important protein in recycling Rabs from their off state in the targeting membrane back to the donor membrane and the on state. The 55-kD guanine nucleotide dissociation inhibitor expressed from SF9 cells has been refined with high-resolution 1.04-Å data collected at the Stanford Synchrotron Radiation Laboratory in collaboration with P. Kuhn. Data for Rab3A have also been obtained to 3.2-Å resolution. The structures of guanine nucleotide dissociation inhibitor, Rab3A, and their complex will reveal the molecular mechanism of how Rabs are regulated between the molecular switch. This project is a collaboration with W. Balch, Department of Molecular Biology.

TISSUE FACTOR

The clinical consequences of thrombosis, including myocardial infarction, deep vein thrombosis, pulmonary embolism, and stroke, are the leading cause of mortality in the industrialized world. The blood coagulation cascade is triggered when plasma is exposed, upon damage to the vasculature, to tissue factor (TF), a cell-surface transmembrane receptor. A potent monoclonal antibody, 5G9, can completely arrest the TF-initiated coagulation cascade, both in vitro and in vivo. 5G9 interrupts an early stage of the coagulation cascade by binding to the complex formed by TF and factor VIIa. This binding prevents (1) formation of a transient ternary complex that consists of TF, factor VIIa, and factor X and (2) subsequent activation of the substrates of the ternary complex, factors IX and X. In collaboration with T. Edgington and W. Ruf, Department of Immunology, we determined the crystal structures of TF, the Fab fragment of 5G9, and the TF-5G9 complex and are pursuing the structure of the TF-VIIa-X ternary complex.

PROTEIN PHOSPHATASE 2A

Protein phosphatase 2A is the most abundant serine/threonine phosphatase in mammalian cells and is important in a number of cellular processes such as cell division, development, and signal transduction. The dimeric core enzyme is composed of a catalytic C subunit and a regulatory A subunit that at times is bound by a member of a family of regulatory B subunits. Protein phosphatase 2A also forms complexes with the small DNA tumor viruses SV40 and polyoma and is involved in tumorigenesis. In collaboration with G. Walter, University of California, San Diego, we have crystallized the A subunit of the phosphatase and collected data to 2.81-Å resolution.

ERYTHROPOIETIN RECEPTOR

Erythropoietin, a 34-kD glycoprotein, is the primary hormone that regulates the differentiation and proliferation of immature erythroid cells. Erythropoietin functions through binding to its receptor on the surface of committed progenitor cells in bone marrow and other hematopoietic tissues. The crystal structures of the extracellular domain of the receptor in complex with both agonist and antagonist peptides have been determined. On the basis of the structural and chemical knowledge derived from the structure of the complex with the agonist, we are pursuing a multidisciplinary drug discovery effort to detect novel erythropoietin agonists and antagonists. This work is being done in collaboration with L. Jolliffe, R.W. Johnson Pharmaceutical Research Institute, and D. Boger, Department of Chemistry. The exciting finding that antagonist peptides dimerize the receptor but do not signal caused us to reevaluate the nature and role of dimerization in signal transduction and cell proliferation.

CANCER TARGETS

Glycinamide ribonucleotide transformylase and aminoimidazole carboxamide ribonucleotide transformylase are folate-dependent enzymes in the de novo biosynthesis of purines and are potential targets for anticancer and antiinflammatory drugs. Work is in progress on a number of folate and nonfolate inhibitors synthesized in D. Boger's laboratory and cocrystallized with wild-type Escherichia coli glycinamide ribonucleotide transformylase. These x-ray structures will guide the development of novel reagents specific for this transformylase that can reduce nonspecific interaction with other folate-dependent enzymes in the cell. This project is also a collaboration with S. Benkovic, Pennsylvania State University.

PUBLICATIONS

Barbas, C.F. III, Heine, A., Zhong, G., Hoffmann, T., Gramatikova, S., Bjornestedt, R., List, B., Anderson, J., Stura, E.A., Wilson, I.A., Lerner, R.A. Immune versus natural selection: Antibody aldolases with enzymic rates but broader scope. Science 278:2085, 1997.

Garcia, K.C., Degano, M., Pease, L.R., Huang, M., Peterson, P.A., Teyton, L., Wilson, I.A. Structural basis of plasticity in T-cell receptor recognition of a self peptide-MHC antigen. Science 279:1166, 1998.

Garcia, K.C., Tallquist, M.D., Pease, L.R., Brunmark, A., Scott, C.A., Degano, M., Stura, E.A., Peterson, P.A., Wilson, I.A., Teyton, L. /alphapub/ß T-cell receptor interactions with syngeneic and allogeneic ligands: Affinity measurements and crystallization. Proc. Natl. Acad. Sci. U.S.A. 94:13838, 1997.

He, M., Gani, M., Livnah, O., Stura, E.A., Beale, D., Coley, J., Wilson, I.A., Taussig, M.J. Sequence, specificity and crystallization of an oestrone-3-glucuronide antibody (3910). Immunology 90:632, 1997.

Heine, A., Stura, E.A., Yli-Kauhaluoma, J.T., Gao, C., Deng, Q., Beno, B.R., Houk, K.N., Janda, K.D., Wilson, I.A. An antibody exo Diels-Alderase inhibitor complex at 1.95-Å resolution. Science 279:1934, 1998.

Huang, M., Syed, R., Stura, E.A., Stone, M.J., Stefanko, R.S., Ruf, W., Edgington, T.S., Wilson, I.A. The mechanism of an inhibitory antibody on TF-initiated blood coagulation revealed by the crystal structures of human tissue factor, Fab 5G9 and TF.G9 complex. J. Mol. Biol. 275:873, 1998.

Johnson, D.L., Farrell, F.X., Barbone, F.P., McMahon, F.J., Tullai, J., Hoey, K., Livnah, O., Wrighton, N.C., Middleton, S.A., Loughney, D.A., Stura, E.A., Dower, W.J., Mulcahy, L.S., Wilson, I.A., Jolliffe, L.K. Identification of a 13 amino acid peptide mimetic of erythropoietin and description of amino acids critical for the mimetic activity of EMP1. Biochemistry 37:3699, 1998.

Scott, C.A., Garcia, K.C., Stura, E.A., Peterson, P.A., Wilson, I.A., Teyton, L. Engineering protein for x-ray crystallography: The murine major histocompatibility complex class II molecule I-A^d. Protein Sci. 7:413, 1998.

Scott, C.A., Peterson, P.A., Teyton, L., Wilson, I.A. Crystal structures of two I-A^d-peptide complexes reveal that high affinity can be achieved without large anchor residues. Immunity 8:319, 1998.

Wilson, I.A., Bjorkman, P.J. Unusual MHC-like molecules: CD1, Fc receptor, the hemochromatosis gene product, and viral homologs. Curr. Opin. Immunol. 10:67, 1998.

Wilson, I.A., Garcia, K.C. T-cell receptor structure and TCR complexes. Curr. Opin. Struct. Biol. 7:839, 1997.