News and Publications

The TCR is a heterodimeric glycoprotein expressed on the surface of T lymphocytes that plays a central role in the recognition of peptide antigens in the context of the MHC. The ability to discriminate between self and foreign antigens depends on the fine specificity of the interaction between TCRs and MHC molecules. We have determined the three-dimensional structure of the murine class I TCR 2C to a resolution of 2.5 Å. Overall, the structure generally resembles an Fab fragment of an antibody. However, significant differences are found in the pairing of the variable and the constant domains and in the unconventional fold of the constant domain of the -chain. The hypervariable loops form a flat surface with a deep central pocket that is involved in the recognition of the peptide antigen bound to the MHC molecule.

To define the molecular basis of the TCR-MHC interaction, we have crystallized and are determining the structures of 2C-MHC complexes with both self and foreign peptide antigens bound to the MHC. The peptide residues contribute about 25% of the surface buried in the formation of the complex. The third complementarity-determining regions of the - and ß-chains of the TCR interact with the central residue of the peptide bound to the MHC. Specific recognition of the MHC molecule is achieved through contacts of complementarity-determining regions 1 and 2 of the - and the ß-chain of the TCR to the helices of the peptide-binding domain.

In the MHC class II system, our focus has been on the production and crystallization of TCRs specific for the murine class II MHC molecule IA^d in complex with ovalbumin, hemagglutinin, and insulin peptides. Structural studies of class II TCR-MHC complexes would address the important question of whether TCR recognition of class I MHC molecules differs substantially from TCR recognition of class II molecules. The TCR-MHC studies are being done in collaboration with L. Teyton, Department of Immunology.

CLASS I AND CLASS II MHC MOLECULES

Several cell-surface molecules involved in T-cell activation have been expressed in soluble forms in collaboration with Dr. Teyton and A. Brunmark, R.W. Johnson Pharmaceutical Research Institute. These include murine MHC class I molecules H-2L^d, H-2K^b mutants and murine class II molecule IA^d, each complexed with peptides. Evaluation of a number of protein engineering strategies was necessary to produce milligram quantities of IA^d for structure-function studies.

H-2L^d is alloreactive with the H-2K^b--specific TCR 2C, and therefore a structural comparison between H-2L^d and H-2K^b is of great value. The structure of H-2L^d has been determined with a mixture of peptides (Fig. 1). The results show that the allogeneic reaction appears to be due mainly to the C-terminal residues of specific peptides that provide the optimal shape and chemistry for 2C reactivity normally present in the syngeneic H-2K^b interaction. The class II molecule IA^d is biologically well characterized and is a factor in murine models of autoimmunity. Two crystal structures of IA^d with different peptides have been determined at 2.4- and 2.6-Å resolution and reveal novel elements of this particular class II peptide binding.

Other unusual interactions between MHC class I molecules and small hapten antigens were examined by determining the structure of H-2K^b complexed with a glycopeptide. The disaccharide of the ligand is visible on the outer boundaries of the binding groove, where interactions with TCRs would predominate. These studies have furnished detailed new insights on antigen selection and presentation to TCRs and have advanced our understanding of these complex interactions that are critical to cellular immunity.

CATALYTIC ANTIBODIES

The 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 (Fig. 2).
Interactions between antibody side chains and the inhibitor provide the basis for understanding the chemical transformation. Modeling studies with transition-state analogs and substrates suggest hypotheses for the preference for the exo vs the endo pathway.

The structure of 33F12, an aldolase antibody that was generated by reactive immunization, has been determined at 2.15-Å resolution. The particular sequence of residues in the binding pocket and the hydrophobic environment of the pocket can account for the unusual pK_a (~6.0) and reactivity of a lysine in the active site. Determination of the structure of a similar natural aldolase enzyme is in progress.

Antibody 5C8 catalyzes the disfavored 6-endo-tet ring closure reaction of trans-epoxy alcohols (violating "Baldwin's rules"). The crystal structures of the Fab fragment of 5C8, unbound (to 2.5 Å) and in complex with the transition-state analog (to 2.0 Å), have been determined (Fig. 3). Further crystallographic and modeling studies are being done to establish the catalytic mechanism and explain the observed regiospecificity and stereospecificity.

The Fab fragments of two antibodies that catalyze another Diels-Alder reaction and a decarboxylation reaction have been crystallized both in their native forms and in complex with their corresponding transition-state analogs. The x-ray data for these two Fabs in both native and complex forms were collected at 2.1 and 2.2 Å, respectively, for each antibody. Refinements of these four structures are in progress. The catalytic antibody projects are being done in collaboration with R. Lerner of the Departments of Chemistry and Molecular Biology; with C. Barbas and C. Shevlin of the Department of Molecular Biology; and with K. Janda, D. Hilvert, and C.-H. Wong of the Department of Chemistry.

NEUTRALIZING ANTIBODIES TO HIV TYPE I

We are studying several antibodies that neutralize HIV type 1. Four of these antibodies were generated by Repligen Corporation (Needham, MA) against a peptide of 40 amino acids that corresponds to the third hypervariable (V3) loop of the HIV type 1 surface glycoprotein gp120. These four antibodies differ in their viral-strain specificity and have overlapping but nonidentical epitopes on the V3 loop. We have determined the structures for Fab fragments of antibody 50.1 in the unliganded and peptide-bound forms, of antibody 59.1 in two peptide-bound forms, and of antibody 58.2 in several peptide-bound forms.

The structures of the peptides bound to the Fabs of 50.1 and 59.1 show that the V3 loop adopts an extended structure, followed by a type II ß-turn around residues Gly-Pro-Gly-Arg. The type II turn is followed by a turn of 3₁₀ helix, resulting in a double turn at the tip of this loop. We replaced an alanine residue in the double-turn region of the loop with an -aminoisobutyric acid residue to constrain the secondary structure in this part of the peptide. The crystal structure of Fab 59.1 in complex with the designed peptide containing the -aminoisobutyric acid residue showed no difference, as expected, from the original complex. However, nuclear magnetic resonance studies by H. Dyson, Department of Molecular Biology, of the original and the designed peptides showed that the -aminoisobutyric acid residue does indeed confer additional structure on the free peptide in solution.

The structures of the Fab of 58.2 in complex with linear and cyclic peptides and with peptides containing -aminoisobutyric acid show that the V3 peptide can adopt a conformation that differs from that of the V3 peptide bound to Fabs of 50.1 and 59.1. In the 58.2 complexes, the peptide differs around residues Gly-Pro-Gly-Arg, adopting a type I turn, followed again by another unclassified turn to form a different type of double turn. The linear and cyclic peptides and the peptides containing -aminoisobutyric acid all adopt this alternative conformation. These results indicate that the V3 loop can adopt at least two conformations on the viral surface.

Antibodies b12 and 3B3 are neutralizing antibodies that recognize the CD4 binding site of gp120. These antibodies were developed in the laboratories of C. Barbas and D. Burton, Department of Molecular Biology. Phage display technology was used to obtain the antibodies from a library of antibodies derived from sera from patients with long-term asymptomatic HIV infection. These antibodies potently neutralize more than 75% of American HIV isolates and 50% of foreign isolates tested and strongly neutralize primary viral isolates. Studies to obtain crystals of the Fab fragments of these antibodies in complex with gp120 are under way. In a collaboration with Progenics (Tarrytown, NY), we are also studying complexes of various CD4 constructs with gp120.

ANTISTEROID ANTIBODIES

Refinement is in progress for a complex of testosterone with an Fab. A molecular replacement solution has also been obtained for the free Fab. The refinement of the Fab to estrone-3-glucuronide is nearing completion.

TISSUE FACTOR

The blood coagulation protease cascade initiated by tissue factor (TF) can be greatly inhibited in vivo by 5G9, a potent monoclonal antibody to human TF. This antibody binds the carboxyl module of the extracellular domain of TF at a nanomolar binding constant and inhibits formation of the ternary complex consisting of TF, factor VIIa, and factor X that is essential for the initiation of the protease cascade in blood coagulation.

To study recognition of TF by the antibody, we determined the crystal structures of the extracellular modules of human TF, an Fab fragment of 5G9, and a complex consisting of TF and the Fab at 2.4, 2.5, and 3.0 Å, respectively, and measured the binding constants of a panel of TF mutants with 5G9 in collaboration with T. Edgington and W. Ruf, Department of Immunology. The structure of the Fab complexed with TF (Fig. 4) explains and confirms the mutagenesis results, illustrates the molecular mechanism of antibody-antigen recognition, and provides insights into the mechanism by which 5G9 can inhibit formation of the ternary complex.

IL-2 RECEPTOR

IL-2 is one of the most important regulators of the immune response. It produces its effects by binding to its receptor, which is composed of three distinct chains, , ß, and . We are involved in crystallographic studies of this heterotrimeric IL-2 receptor system in a collaboration with T. Ciardelli at Dartmouth College.

ERYTHROPOIETIN RECEPTOR

The structure of the extracellular domain of the receptor for erythropoietin and an antagonist peptide has been determined to the resolution of 2.8 Å. The antagonist peptide was derived from a set of agonist peptides, which were discovered via phage-display techniques in collaboration with R.W. Johnson Pharmaceutical Research Institute and Affymax, Palo Alto, California. A single amino acid modification (tyrosine to 3,5-dibromotyrosine) reversed the activation properties of the peptide. The structure reveals a 2:2 assembly in which two peptides bind two receptor molecules and generate an asymmetric receptor dimerization, a condition that differs from the perfect twofold symmetry observed for the complex containing the peptide agonist.

Symmetric dimerization is a common feature of other erythropoietin receptor--peptide complexes that have agonistic properties. It is well established that in many systems receptor dimerization is the initial key event in signaling, but in this instance, we showed that a nonproductive mode of dimerization occurs. Such a difference in dimer formation can have a great impact on the understanding of signaling in both cytokine and other receptor systems. We are currently determining structures of the agonist peptide complexes with the erythropoietin receptor in collaboration with L. Jolliffe, R.W. Johnson Pharmaceutical Research Institute.

CANCER TARGETS

Glycinamide ribonucleotide transformylase and aminoimidazole carboxamide ribonucleotide transformylase are folate-dependent enzymes involved in the de novo biosynthesis of purine. These enzymes are potential targets for anticancer and antiinflammatory drugs. Crystal structures are in progress for a number of folate-derived inhibitors synthesized in D. Boger's laboratory, Department of Chemistry, and cocrystallized with wild-type Escherichia coli glycinamide ribonucleotide transformylase. These x-ray structures will guide future development of novel nonfolate analogs specific for glycinamide ribonucleotide transformylase, thereby reducing nonspecific interaction with other folate-dependent enzymes in the cell.

Recently, a novel mutant form of E. coli glycinamide ribonucleotide transformylase that lacks the ability to form dimers was developed by S. Benkovic, Pennsylvania State University, in conjunction with P. Jenning's laboratory, University of California, San Diego. We are determining the x-ray structures of this mutant and of additional nonactive mutant structures of glycinamide ribonucleotide transformylase in a number of complexed forms to provide evidence for the mechanism of transformylation.

GUANINE NUCLEOTIDE DISSOCIATION INHIBITOR

The guanine nucleotide dissociation inhibitor (GDI) regulates the retrieval of Rabs in the vesicular traffic while inhibiting the dissociation of GDP from Rabs, small GTP-binding proteins related to the Ras superfamily. The crystal structure of the bovine -isoform of GDI expressed from E. coli has been determined to 1.8-Å resolution. Mutagenesis studies have shown that a substructure in domain I is possibly involved in Rab association. The structure of the GDI-Rab complex has also been investigated. Meanwhile, a more active form of GDI expressed from insect cells has been crystallized, and x-ray data have been collected at 1.7-Å resolution. The high-resolution structure of this GDI will reveal any posttranslational modifications that this molecule undergoes in higher eukaryotic systems.

CD1

CD1 is an MHC-like antigen-presenting molecule. Recent studies have shown that CD1 may present mycobacterial cell wall lipids or phage display--derived peptides as antigens to T cells. Attempts to elute a natural ligand directly from CD1 molecules have been unsuccessful. The crystal structure of mouse CD1d1 has been determined to 2.8-Å resolution in two different space groups. Determination of the structure revealed a molecule markedly similar to MHC class I molecules but with a larger, mostly hydrophobic, ligand-binding groove (Fig. 5).

A second structure determination was undertaken to elucidate the nature of the interaction between CD1 and a large hydrophobic peptide presented to T cells by mouse CD1d1. Several features of the molecule are now apparent that were poorly ordered in the original crystal structure, including several carbohydrate moieties. Surprisingly, a long unbranched ligand occupying the binding groove is also apparent. The density for this ligand is consistent with density seen in the binding groove from the original structure determination and is not consistent with peptide. Attempts to crystallize the CD1d1-peptide complex are continuing. The studies on mouse CD1d1 have been done in collaboration with P. Peterson's group and others at R.W. Johnson Pharmaceutical Research Institute; those on human CD1, with M. Kronenberg, La Jolla Institute of Allergy and Immunology.

We are also attempting to crystallize the human CD1b isoform. Earlier reports stated that this molecule presents mycolic acid derived from mycobacterial extracts to T lymphocytes. Recent studies detected a series of natural lipoglycans that are ligands for human CD1b and that elicit T-cell responses. The structure of the complex of CD1b with a lipid would provide insights into the presentation of nonpeptide antigens by MHC-like molecules to T cells. The studies on CD1b have been done in collaboration with R. Modlin, University of California, Los Angeles, with ligands provided by M. Brenner and S. Porcelli, Harvard Medical School.

Top of Page

Methods for the Crystallization of Macromolecules

E.A. Stura, S. Ghosh, A. Muhlberg, K. Hotta, P. Shim

The development of methods and techniques for the rapid determination of crystallization conditions with minimal amounts of proteins and nucleic acids has been the focus of the research this current year.

Reverse screening, a method based on the principle that a precipitant or buffer should play no role other than that of inducing supersaturation or maintaining the pH, has been applied to proteins that have a binding site for phosphate moieties. In collaboration with J. Elder and C. Stout, Department of Molecular Biology, we used sodium citrate instead of ammonium sulfate, which is a phosphate mimic, to obtain complex crystals of dUTPase with deoxynucleotide diphosphates and triphosphates. These complexes are not compatible with the 1.8-Å diffracting crystals obtained by using cacodylate, another phosphate mimic, which can accommodate only deoxynucleotide monophosphates.

Reverse screening has been also applied to the crystallization of the humanized catalytic antibody Hu21D8. Crystals of the native antibody were obtained by slightly changing the conditions used for the crystallization of Fab fragments of catalytic antibodies obtained in the P.G. Schultz laboratory at the University of California, Berkeley, by using a similar expression and purification system. In collaboration with C. Stout and W. Chazin, Department of Molecular Biology, we are applying the principles of reverse screening to crystallization of RNA and RNA-DNA enzymes with good success.

Dynamic light scattering can be used to determine the aggregation state and polydispersity of macromolecules. Polydispersity can be considered a measure of heterogeneity and, to a certain extent, of the likelihood of crystallization. This technique has been used to differentiate the dimeric and monomeric states of the erythropoietin receptor in the presence of different ligands and to confirm the trimeric state of dUTPase of feline immunodeficiency virus. In collaboration with S. Schmid, Department of Molecular Biology, we have determined conditions in which dynamin remains relatively monodisperse in its tetrameric form.

Initial crystals of gap junctions have been obtained in collaboration with B. Gilula, Department of Cell Biology.

PUBLICATIONS

Cabezas, E., Stanfield, R.L., Wilson, I.A., Satterthwait, A.C. Defining conformational requirements for the principle neutralizing determinant on HIV-1. In: Peptides: Chemistry, Structure and Biology. Proceedings of the 14th American Peptide Symposium. Kaumaya, P. (Ed.). ESCOM, Leiden, the Netherlands, 1996, p. 800.

Desmet, J., Wilson, I.A., Joniau, M., De Maeyer, M., Lasters, I. Computation of the binding of fully flexible peptides to proteins with flexible side chains. FASEB J. 11:164, 1997.

Garcia, K.C., Scott, C.A., Brunmark, A., Carbone, F.R., Peterson, P.A., Wilson, I.A., Teyton, L. CD8 enhances formation of stable T-cell receptor/MHC class I molecule complexes. Nature 384:577, 1996.

Ghiara, J.B., Ferguson, D.C., Satterthwait, A.C., Dyson, H.J., Wilson, I.A. Structure-based design of a constrained peptide mimic of the HIV-1 V3 loop neutralization site. J. Mol. Biol. 266:31, 1997.

Haynes, M.R., Heine, A., Wilson, I.A. Catalytic antibody structures: An early assessment. Isr. J. Chem. 36:133, 1996.

Haynes, M.R., Lenz, M., Taussig, M.J., Wilson, I.A., Hilvert, D. Sequence similarity and cross-reactivity of a Diels-Alder catalyst and an anti-progesterone antibody. Isr. J. Chem. 36:151, 1996.

Stura, E.A., Ruf, W., Wilson, I.A. Crystallization and preliminary crystallographic data for a ternary complex between tissue factor, factor VIIa, and a BPTI-derived inhibitor. J. Cryst. Growth 168:260, 1996.

White J.M., Hoffman, L.R., Arevalo, J.H., Wilson, I.A. Attachment and entry of influenza virus into host cells: Pivotal roles of the hemagglutinin. In: Structural Biology of Viruses. Chiu, W., Burnett, R.M., Garcea, R. (Eds.). Oxford Press, New York, 1997, p. 80.

Wu, S.K., Zeng, K., Wilson, I.A., Balch, W.E. Structural insights into the function of the Rab GDI superfamily. Trends Biochem. Sci. 21:472, 1996.

Zeng, Z.H., Castano, A.R., Segelke, B.W., Stura, E.A., Peterson, P.A., Wilson,I.A. Crystal structure of mouse CD1: An MHC-like fold with a large hydrophobic binding groove. Science, in press.

Top of Page

Principles of Protein Structure for Recognition, Interaction, and Function