Scientific Report 2004

Molecular Biology

Crystallographic Studies of Immune Recognition, Molecular Assemblies, and Anticancer Targets

I.A. Wilson, R.L. Stanfield, Y. An, T.A. Bowden, D.A. Calarese, R.M.F. Cardoso, J.-W. Choe, A.L. Corper, M.D.M. Crispin, T.H. Cross, X. Dai, W.L. Densley, E.W. Debler, M.-A. Elsliger, S. Ferguson, P.A. Horton, S. Ito, M.S. Kelker, J.G. Luz, J.B. Reiser, E.B. Shillington, D.A. Shore, D.J. Stauber, R.S. Stefanko, J. Stevens, P. Verdino, D.W. Wolan, L. Xu, M. Yu, D.M. Zajonc, Y. Zhang, X. Zhu

We are investigating many different families of immune recognition receptors and anticancer targets. We use primarily x-ray crystallography to determine structures of these biomedically relevant proteins in complex with their respective ligands or inhibitors. Structural information is used in some projects for design of novel compounds to bind or inhibit the proteins of interest. Our overall goal is to understand how foreign pathogens are recognized by the host adaptive and innate immune systems.

Viral Coat Proteins

In 1918–1919, the great influenza pandemic (Spanish flu) killed an estimated 40 million persons. In collaboration with J. Taubenberger, Armed Forces Institute of Pathology, Washington, DC, and P. Palese, A. García-Sastre, and C. Basler, Mount Sinai School of Medicine, New York, New York, we are carrying out structural analyses of the 1918 viral proteins to understand why this strain was so pathogenic. Our first structure determined was of hemagglutinin, the major surface glycoprotein that is involved in binding and fusion of influenza virus to human respiratory cells (Fig. 1). The structure reveals features conserved within avian viruses that may have contributed to the increased virulence of this virus.

Fig 1. Ribbon representation of the hemagglutinin HA0 trimer from the 1918 influenza virus. Each monomer has 2 important sites: the receptor-binding site for virus attachment to the host lung epithelial cells via sialic acid–containing host cell receptors and the cleavage site where for full infectivity, the single chain (HA0) is cut intousion peptide that is critical for subsequent membrane fusion events that lead to infection.

HIV Type 1 Neutralizing Antibodies

The HIV type 1 (HIV-1) envelope glycoproteins gp120 and gp41 mutate rapidly in response to antibody challenge, thus allowing the virus to evade the immune system. However, a few rare antibodies can neutralize primary strains of HIV-1. The crystal structures of the Fabs of the broadly neutralizing antibodies 4E10 with a gp41 peptide (Fig. 2), 2G12 with its carbohydrate epitope, and 447-52D with the gp120 V3 loop have revealed key epitope conformations that are being used as templates for rational design of HIV-1 vaccines.

Fig 2. Antigen-binding site of the Fab of 4E10, a broadly neutralizing antibody to HIV-1. This Fab recognizes a helical epitope from the gp41 glycoprotein. The conformations of 4E10 complementarity-determining regions L1, L2, L3, H1, H2, and H3 and the bound helical gp41 epitope are shown.

The research on HIV is 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 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; R. Wyatt and P. Kwong, Vaccine Research Center, National Institutes of Health, Bethesda, Maryland; and the International Aids Vaccine Initiative.

Peptidoglycan Recognition

Peptidoglycans, essential components of the cell walls of all bacteria, are among the conserved motifs that invoke the innate immune system to induce strong antibacterial responses. A family of pattern recognition molecules, peptidoglycan recognition proteins (PGRPs), which interact with peptidoglycans, can be functionally divided into “catalytic” PGRPs that have amidase activity and “recognition” PGRPs that bind peptidoglycans. We determined the 1.56-Å crystal structure of PGRP-SA (Fig. 3), which does not contain the canonical zinc found in catalytic PGRPs. Comparison of PGRP-SA with the catalytic PGRP-LB indicated overall structural conservation and a hydrophilic groove that most likely is the peptidoglycan core binding site. These investigations were done in collaboration with L. Teyton, Department of Immunology.

Fig. 3. The 3-dimensional ribbon structure of PGRP-SA from Drosophila melanogaster. The residues and atoms that line the binding groove and that may be involved in the interactions with the bacterial peptidoglycan core are shown in a ball-and-stick representation.

Triggering Receptor Family

The triggering receptor expressed on myeloid cells (TREM) family of extracellular immunoglobulin receptors includes both activating and inhibitory isoforms whose ligands are unknown. TREM-1 amplifies the inflammation induced by both bacteria and fungi and is a potential therapeutic target. The 1.47-Å structure of the extracellular domain of human TREM-1, coupled with analytical ultracentrifugation and deuterium-hydrogen nuclear magnetic resonance spectroscopy of both human and mouse TREM-1, conclusively showed the monomeric state of this extracellular ectodomain in solution. This research was also done collaboration with Dr. Teyton.

Catalytic Antibodies

Development of effective immunotherapy for cocaine abuse, addiction, and overdose is under way. In studies done in collaboration with P. Wirsching and K.D. Janda, Department of Chemistry, the crystal structures of cocaine-hydrolyzing antibodies 7A1 (free, with cocaine, with transition-state analog, and with products) and 3A6 (with cocaine and with products) delineated the major steps along the reaction coordinate.

Antibodies can catalyze the generation of hydrogen peroxide from singlet dioxygen and water via the postulated intermediate dihydrogen trioxide and other trioxygen species. In studies with R.A. Lerner and P. Wentworth, Department of Chemistry, we used x-ray analyses of catalytic antibodies 4C6 and 13G5 to elucidate the chemical consequences to the antibody molecule of exposure to such reactive intermediates. The results suggested locations on the antibody where these intermediate species could be generated.

Antibody 34E4 catalyzes the base-promoted E2 elimination of substituted benzisoxazoles and is one of the most efficient abzymes characterized to date. Structures of the Fab of 34E4 reveal a deep binding pocket with high shape complementarity to the transition-state analog and strong hydrophobicity provided by aromatic residues. Glu^H50 interrupts this hydrophobic belt and could act as a general base to abstract a proton from the substrate, triggering elimination of the phenoxide. This research is being done in collaboration with D. Hilvert, ETH Zürich, Zürich, Switzerland.

Classical and Nonclassical MHC Molecules

CD1 molecules, nonclassical homologs of class I MHC molecules, present lipid antigens to CD1-restricted T-cell receptors (TCRs). Recently, mycobacterial lipopeptide intermediates of the siderophore biosynthetic pathway were identified as CD1a antigens. The 2.8-Å structure of CD1a in complex with a synthetic lipopeptide derivative (Fig. 4) revealed that the lipid part of the ligand is buried deep within the binding groove, whereas the peptidic headgroup is exposed at the surface for recognition by specific CD8⁺ TCRs.

Fig. 4. Antigen-binding groove of CD1a. The molecular surface is shown as a transparent binding pocket with the bound lipopeptide ligand. The side view (B) shows both the deeply buried lipid part and the peptidic headgroup, which is more exposed and accessible by the TCR (top view, A).

Ligands are provided by our collaborators D.B. Moody and M.B. Brenner, Harvard Medical School, Boston, Massachusetts, and C.-H. Wong, Department of Chemistry. Collaborators in the research on CD1 and TCRs include M. Kronenberg, La Jolla Institute for Allergy and Immunology, San Diego, California; V. Kumar, Torrey Pines Institute for Molecular Studies, San Diego, California; Wayne Severn, AgResearch, Upper Hutt, New Zealand; and R. Dwek, P. Rudd, and S. Davis, University of Oxford, Oxford, England.

MUC1, a cancer mucin, is a promising target for the development of an anticancer vaccine. In collaboration with V. Apostolopoulos, Austin Research Institute, Heidelberg, Australia, we are determining structures for a number of murine H2-K^b class I MHC molecules with MUC1-derived peptides to explain how noncanonical, low-affinity peptides can stimulate cytotoxic T lymphocytes. Structural comparisons between glycosylated and nonglycosylated peptides will facilitate design of novel peptide mimics as tumor vaccines.

The TCR coreceptor CD8 is expressed on the surface of leukocyte cells as an αβ heterodimer and an αα homodimer; the former is the dominant isotype on circulatory CD8⁺ cytotoxic T cells. Large quantities of soluble CD8 αα and αβ have been produced for crystallization, and CD8 αα has been crystallized in complex with the Fab YTS in collaboration with Dr. Teyton.

Cytokine Receptors

IL-2, a class I cytokine, functions as a growth factor in the immune system, causing proliferation and cytokine production in T cells, proliferation and antibody production in B cells, activation of the cytotoxic activity of natural killer cells, and proliferation and clonal expansion of tumor-specific T cells. IL-2 signaling occurs through a ligand-induced activation of the high-affinity heterotrimeric IL-2 receptor (α-, β-, and γ-chains) complex. The structure of this complex will provide insight into the recognition, assembly, and signaling properties of the IL-2 receptor and will aid in design of novel ligands to modulate function and activity of the receptor. Our collaborator in this research is K. Smith, Weil Medical College of Cornell University, New York, New York.

Enzymatic Cancer Targets

Rapidly dividing cells are critically dependent on de novo nucleotide synthesis pathways, essential for DNA synthesis and repair. This vulnerability has long been exploited in the area of anticancer research, and antifolates are one of the most extensively investigated classes of antineoplastic agents. Our focus involves 2 enzymes in the de novo purine biosynthesis pathway: glycinamide ribonulceotide transformylase (Fig. 5) and the bifunctional aminoimidazole carboxamide ribonucleotide transformylase inosine monophosphate cyclohydrolase.

Fig. 5. Stereoview of the active binding site of human glycinamide ribonucleotide (GAR) transformylase in complex with a folate analog, 10-trifluoroacetyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-CF3CO-DDACTHF), and substrate β-GAR. The final refined structure of 10-CF3CO-DDACTHF is superimposed on the 2Fo-Fc electron density contoured at 2 σ, and the substrate β-GAR structure is shown within the 2Fo-Fc electron density contoured at 1.5 σ.

The structures of these 2 enzymes in complex with several different compounds revealed the mechanism of the formyl transfer reaction and provided a platform for the design of inhibitors to develop antineoplastic agents. These investigations are being done in collaboration with D. Boger, Department of Chemistry; G.P. Beardsley, Yale University, New Haven, Connecticut; and S.J. Benkovic, Pennsylvania State University, University Park, Pennsylvania.

Publications

Burton, D.R., Desrosiers, R.C., Doms, R.W., Koff, W.C., Kwong, P.D., Moore, J.P., Nabel, G.J., Sodroski, J., Wilson, I.A., Wyatt, R.T. HIV vaccine design and the neutralizing antibody problem. Nat. Immunol. 5:233, 2004.

Cheong, C.G., Wolan, D.W., Greasley, S.E., Horton, P.A., Beardsley, G.P., Wilson, I.A. Crystal structures of human bifunctional enzyme aminoimidazole-4-carboxamide ribonucleotide transformylase/IMP cyclohydrolase in complex with potent sulfonyl-containing antifolates. J. Biol. Chem. 279:18034, 2004.

Desharnais, J., Hwang, I., Zhang, Y., Tavassoli, A., Baboval, J., Benkovic, S.J., Wilson, I.A., Boger, D.L. Design, synthesis and biological evaluation of 10-CF₃CO-DDACTHF analogues and derivatives as inhibitors of GAR Tfase and the de novo purine biosynthetic pathway. Bioorg. Med. Chem. 11:4511, 2003.

Erlandsen, H., Canaves, J.M., Elsliger, M.A., et al. Crystal structure of an HEPN domain protein (TM0613) from Thermotoga maritima at 1.75 Å resolution. Proteins 54:806, 2004.

Kuhmann, S.E., Pugach, P., Kunstman, K.J., Taylor, J., Stanfield, R.L., Snyder, A., Strizki, J.M., Riley, J., Baroudy, B.M., Wilson, I.A., Korber, B.T., Wolinsky, S.M., Moore, J.P. Genetic and phenotypic analyses of human immunodeficiency virus type 1 escape from a small-molecule CCR5 inhibitor [published correction appears in J. Virol. 78:6706, 2004]. J. Virol. 78:2790, 2004.

Lee, H.K., Scanlan, C.N., Huang, C.Y., Chang, A.Y., Calarese, D.A., Dwek, R.A., Rudd, P.M., Burton, D.R., Wilson, I.A., Wong, C.H. Reactivity-based one-pot synthesis of oligomannoses: defining antigens recognized by 2G12, a broadly neutralizing anti-HIV-1 antibody. Angew. Chem. Int. Ed. 43:1000, 2004.

Marsilje, T.H., Hedrick, M.P., Desharnais, J., Capps, K., Tavassoli, A., Zhang, Y., Wilson, I.A., Benkovic, S.J., Boger, D.L. 10-(2-benzoxazolcarbonyl)-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid: a potential inhibitor of GAR transformylase and AICAR transformylase. Bioorg. Med. Chem. 11:4503, 2003.

Marsilje, T.H., Hedrick, M.P., Desharnais, J., Tavassoli, A., Zhang, Y., Wilson, I.A., Benkovic, S.J., Boger, D.L. Design, synthesis, and biological evaluation of simplified α-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues as potential inhibitors of GAR transformylase and AICAR transformylase. Bioorg. Med. Chem. 11:4487, 2003.

Mitra, A.K., Celia, H., Ren, G., Luz, J.G., Wilson, I.A., Teyton, L. Supine orientation of a murine MHC class I molecule on the membrane bilayer. Curr. Biol. 14:718, 2004.

Moody, D.B., Young, D.C., Cheng, T.Y., Rosat, J.P., Roura-Mir, C., O’Connor, P.B., Zajonc, D.M., Walz, A., Miller, M.J., Levery, S.B., Wilson, I.A., Costello, C.E., Brenner, M.B. T cell activation by lipopeptide antigens [published correction appears in Science 304:211, 2004]. Science 303:527, 2004.

Page, R., Nelson, M.S., von Delft, F., et al. Crystal structure of -glutamyl phosphate reductase (TM0293) from Thermotoga maritima at 2.0 Å resolution. Proteins 54:157, 2004.

Pantazatos, D., Kim, J.S., Klock, H.E., Stevens, R.C., Wilson, I.A., Lesley, S.A., Woods, V.L., Jr. Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS. Proc. Natl. Acad. Sci. U. S. A. 101:751, 2004.

Pantophlet, R., Wilson, I.A., Burton, D.R. Hyperglycosylated mutants of human immunodeficiency virus (HIV) type 1 monomeric gp120 as novel antigens for HIV vaccine design. J. Virol. 77:5889, 2003.

Rader, C., Turner, J.M., Heine, A., Shabat, D., Sinha, S.C., Wilson, I.A., Lerner, R.A., Barbas, C.F. A humanized aldolase antibody for selective chemotherapy and adaptor immunotherapy. J. Mol. Biol. 332:889, 2003.

Rudolph, M.G., Shen, L.Q., Lamontagne, S.A., Luz, J.G., Delaney, J.R., Ge, Q., Cho, B.K., Palliser, D., McKinley, C.A., Chen, J., Wilson, I.A., Eisen, H.N. A peptide that antagonizes TCR-mediated reactions with both syngeneic and allogeneic agonists: functional and structural aspects. J. Immunol. 172:2994, 2004.

Rudolph, M.G., Wingren, C., Crowley, M.P., Chien, Y.H., Wilson, I.A. Combined pseudo-merohedral twinning, non-crystallographic symmetry and pseudo-translation in a monoclinic crystal form of the γδ T-cell ligand T10. Acta Crystallogr. D Biol. Crystallogr. 60(Pt. 4):656, 2004.

Schwarzenbacher, R., Canaves, J.M., Brinen, L.S., et al. Crystal structure of uronate isomerase (TM0064) from Thermotoga maritima at 2.85 Å resolution. Proteins 53:142, 2003.

Schwarzenbacher, R., Deacon, A.M., Jaroszewski, L., et al. Crystal structure of a putative glutamine amido transferase (TM1158) from Thermotoga maritima at 1.7 Å resolution. Proteins 54:801, 2004.

Schwarzenbacher, R., Jaroszewski, L., von Delft, F., et al. Crystal structure of a phosphoribosylaminoimidazole mutase PurE (TM0446) from Thermotoga maritima at 1.77-Å resolution. Proteins 55:474, 2004.

Schwarzenbacher, R., von Delft, F., Abdubek, P., et al. Crystal structure of a putative PII-like signaling protein (TM0021) from Thermotoga maritima at 2.5 Å resolution. Proteins 54:810, 2004.

Schwarzenbacher, R., von Delft, F., Canaves, J.M., et al. Crystal structure of an iron-containing 1,3-propanediol dehydrogenase (TM0920) from Thermotoga maritima at 1.3 Å resolution. Proteins 54:174, 2004.

Stanfield, R.L., Gorny, M.K., Williams, C., Zolla-Pazner, S., Wilson, I.A. Structural rationale for the broad neutralization of HIV-1 by human monoclonal antibody 447-52D. Structure (Camb.) 12:193, 2004.

Stevens, J., Corper, A.L., Basler, C.F., Taubenberger, J.K., Palese, P., Wilson, I.A. Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenza virus. Science 303:1866, 2004.

Wolan, D.W., Cheong, C.G., Greasley, S.E., Wilson, I.A. Structural insights into the human and avian IMP cyclohydrolase mechanism via crystal structures with the bound XMP inhibitor. Biochemistry 43:1171, 2004.

Wolan, D.W., Greasley, S.E., Wall, M.J., Benkovic, S.J., Wilson, I.A. Structure of avian AICAR transformylase with a multisubstrate adduct inhibitor β-DADF identifies the folate binding site. Biochemistry 42:10904, 2003.

Zajonc, D.M., Elsliger, M.A., Teyton, L., Wilson, I.A. Crystal structure of CD1a in complex with a sulfatide self antigen at a resolution of 2.15 Å. Nat. Immunol. 4:808, 2003.

Zhu, X., Wentworth, P., Jr., Wentworth, A.D., Eschenmoser, A., Lerner, R.A., Wilson, I.A. Probing the antibody-catalyzed water-oxidation pathway at atomic resolution. Proc. Natl. Acad. Sci. U. S. A. 101:2247, 2004.

Zwick, M.B., Komori, H.K., Stanfield, R.L., Church, S., Wang, M., Parren, P.W., Kunert, R., Katinger, H., Wilson, I.A., Burton, D.R. The long third complementarity-determining region of the heavy chain is important in the activity of the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2F5. J. Virol. 78:3155, 2004.

Zwick, M.B., Parren, P.W., Saphire, E.O., Church, S., Wang, M., Scott, J.K., Dawson, P.E., Wilson, I.A., Burton, D.R. Molecular features of the broadly neutralizing immunoglobulin G1 b12 required for recognition of human immunodeficiency virus type 1 gp120. J. Virol. 77:5863, 2003.