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The Skaggs Institute
for Chemical Biology


Crystallographic Studies of Immune Recognition and Therapeutic Targets


I.A. Wilson, K. Beis, D.A. Calarese, R.M.F. Cardoso, P.J. Carney, J.W. Choe, E.W. Debler, M.-A. Elsliger, S. Ferguson, M.J. Jimenez-Dalmaroni, R.L. Stanfield, R.S. Stefanko, J. Stevens, J.A. Vanhnasy, E. Wise, D.W. Wolan, L. Xu, D.M. Zajonc

Detailed crystallographic structural information is valuable for a thorough understanding of how proteins function in molecular processes. The goal of our research is to gain such information on proteins of high interest, including those involved in innate immunity, viral pathogenesis, and cancer chemotherapy. Knowledge gained from these studies is being used in the design of possible vaccines and drugs.

The Innate Immune Response Against Microbial Pathogens

Toll-like receptors (TLRs), a family of mammalian glycoproteins involved in activation of the immune response, recognize conserved structures in bacteria, virus, yeasts, and helminths. We recently determined the crystal structure of the human TLR3 ectodomain to 2.1-Å resolution; the structure shows that the ectodomain folds into a large horseshoe-shaped solenoid assembled from 23 leucine-rich repeat motifs (Fig. 1).

Fig. 1. The TLR3 ectodomain. The 23 leucine-rich repeats form a horseshoe-shaped molecule.

A total of 7 conserved hydrophobic residues in the leucine-rich repeat motif form a tight hydrophobic core, and a conserved asparagine residue at position 10 contributes an extensive hydrogen-bonding network for solenoid stabilization. TLR3 is largely masked by carbohydrate, with 15 predicted N-glycosylation sites; however, one face is glycosylation-free, a finding that could indicate the likely ligand-binding site or oligomerization interface. The inner concave surface of the protein is filled with carbohydrate and is predominantly negatively charged, contradicting any role in RNA binding. The glycosylation-free face contains a surface patch composed of highly conserved residues and an insertion of a TLR3-specific leucine-rich repeat that together form a homodimer interface in the crystal; 2 additional patches of positively charged residues and a second insertion might provide an appropriate binding site for double-stranded RNA.

We are also cloning and expressing other human TLRs, including TLR1, TLR2, and TLR6, and the CD36 coreceptor, to determine their structures and to elucidate their ligand-binding specificity. Understanding these interactions is crucial for determinations of how microorganisms are sensed by the innate immune system and for the design of novel, selective agonists and antagonists of TLR signaling pathways. This work is being done in collaboration with B. Beutler and R. Ulevitch, Scripps Research.

Members of the CD1 family of nonclassical MHC class I antigen receptors present lipid ligands to specific T cells for immune recognition. We recently determined the crystal structures of mouse CD1d in complex with the self-antigen sulfatide (Fig. 2) and in complex with a short-chain α-galactosyl ceramide that is the most stimulatory ligand for natural killer T cells.

Fig. 2. Hydrogen-bond network between mouse CD1d and the sulfatide ligand.

The different linkages between the carbohydrate and the lipid backbone in each ligand result in structural differences in the presentation and orientation of the sugar moieties of the ligands, which are recognized by different subsets of natural killer T cells. This work is a collaboration with M.B. Brenner and D.B. Moody, Harvard Medical School, Boston, Massachusetts; L. Teyton, Scripps Research; C.-H. Wong, the Skaggs Institute; M. Kronenberg, La Jolla Institute of Allergy and Immunology, San Diego, California; V. Kumar, Torrey Pines Institute for Molecular Studies, San Diego, California; and W. Severn and G. Painter, Industrial Research Limited, Lower Hut, New Zealand.

The protein nucleotide-binding oligomerization domain 2 (NOD2) is an important intracellular receptor that recognizes structures present in bacteria called peptidoglycans. Mutations in NOD2 have been associated with the inflammatory Crohn’s disease. In collaboration with R. Ulevitch, Scripps Research, we are working to produce and crystallize the putative binding domain of NOD2 and the NOD2 intracellular domain, which is involved in signal transduction.

HIV Vaccine Design

An effective vaccine against HIV type 1 (HIV-1) must be able to protect against the wide variety of HIV-1 strains that are currently circulating. To date, only a few antibodies have been discovered in patients infected with HIV-1 that can neutralize such a broad range of viral isolates. We have determined crystal structures for several of these broadly neutralizing antibodies in complex with the viral antigens to provide templates for the design of immunogens to elicit similar types of antibody responses.

Human antibody 4E10 neutralizes a broad range of HIV-1 isolates and recognizes a contiguous and highly conserved helical epitope in the membrane-proximal region of the viral envelope protein gp41. To investigate the effect of constraints that promote the formation of helices on the interaction between 4E10 and its helical epitope, we determined the crystal structure of the Fab of 4E10 in complex with 3 different constrained peptides containing the consensus group M 4E10 epitope. The incorporation of α-aminoisobutyric acid and thioether-linked side chains of cysteine and ornithine limits the conformational flexibility of these helical peptides while enhancing the interaction between antibody and epitope. These structures have allowed us to define a minimal consensus motif within the 4E10 epitope for further design efforts. Forcing the peptide to adopt a specific ensemble of functionally relevant conformations should make it possible to elicit neutralizing antibodies via vaccination and thus aid in efforts to design HIV-1 vaccines.

The crystal structure of 2G12, another broadly neutralizing antibody to HIV-1, is also being used as a template for the design of immunogens. 2G12 recognizes a patch of oligomannose carbohydrates on the gp120 viral envelope protein. Several crystal structures of 2G12 with synthetic oligomannose sugars have revealed the basis of the carbohydrate specificity. Testing of these potential carbohydrate immunogens is under way.

The research on HIV-1 is being done in collaboration with D.R. Burton and P.E. Dawson, Scripps Research; C.-H. Wong, the Skaggs Institute; S. Danishefsky, Memorial Sloan-Kettering Cancer Center, New York, New York; and H. Katinger, University of Agriculture, Vienna, Austria.

Influenza Virus Glycoproteins

Influenza is a contagious viral respiratory disease that annually affects 10%–20% of the human population. In recent history, 4 outbreaks reached pandemic proportions, the outbreaks of 1890, 1918, 1957, and 1968. Of these, the 1918 outbreak was the most destructive, killing an estimated 40 million people. As members of a “1918 flu consortium,” we are collaborating with researchers from the Armed Forces Institute of Pathology, Washington, DC; Mount Sinai School of Medicine, New York, New York; University of Washington, Seattle, Washington; and the Centers for Disease Control and Prevention, Atlanta, Georgia; to uncover the reasons why this particular influenza virus was so pathogenic and how it managed to evade the immune system so effectively.

Currently, we are undertaking structural analyses of a number of proteins from the 1918 virus. The first structure to be completed is that of hemagglutinin, the virus surface glycoprotein involved in viral infectivity. Analysis of this structure revealed a number of serotype and avian-like features that may have contributed to its increased virulence. We recently analyzed the receptor specificity of the hemagglutinins from the 1918 virus and from more modern viruses, such as the H5 avian influenza virus that is currently prevalent in Asia, by comparing their binding to a panel of carbohydrates. In collaboration with O. Blixt and J. Paulson, Consortium for Functional Glycomics, La Jolla, California, who recently developed an extraordinarily useful glycan array, we are cloning/expressing a number of hemagglutinins for analysis with this array. For the 2 known 1918 hemagglutinins, a single point mutation is sufficient to switch from exclusively α2,6 to α2,6/α2,3 specificity. We are also investigating whether such a minimal change is all that is required for the current H5 avian influenza hemagglutinins to cross the avian-human species barrier.

Antifolates for Chemotherapy

The enzymes aminoimidazole carboxamide ribonucleotide (AICAR) transformylase inosine monophosphate cyclohydrolase (ATIC) and glycinamide ribonucleotide transformylase are key folate-dependent enzymes in the de novo purine biosynthesis pathway. Cancer cells are highly dependent on this pathway, making these enzymes good targets for antineoplastic chemotherapeutic agents.

ATIC is a homodimeric enzyme that encompasses activities of both AICAR transformylase and inosine monophosphate cyclohydrolase. Structure-based development of specific novel inhibitors that do not interact with other folate-dependent enzymes is in progress in collaborative studies with D.L. Boger, the Skaggs Institute, and S.J. Benkovic, Pennsylvania State University, University Park, Pennsylvania.

Crystal structures of avian ATIC in complex with various substrates and inhibitors have revealed the active-site locations of the AICAR transformylase substrate and cofactor, as well as the binding domain of inosine monophosphate cyclohydrolase. In collaboration with A.J. Olson, Scripps Research, using molecular docking of small molecules from the National Cancer Institute diversity sets, we did a systematic search for novel inhibitors of AICAR transformylase. A total of 44 compounds unrelated to known antifolates were identified, and 16 with good solubility were tested for their ability to inhibit the activity of AICAR transformylase. Remarkably, 8 of these compounds inhibited AICAR transformylase in vitro in micromolar concentrations.

Following up this lead, we identified several more potent inhibitors by doing a similarity search in the National Cancer Institute database and the Available Chemical Directory database. Our results support this combined computational/structural approach for rational design of novel nonfolate inhibitors of AICAR transformylase, and crystal structures of AICAR in complex with the compounds are being determined.

The IL-2 Receptor

The interleukin IL-2 is a cytokine that functions as a T-cell growth factor and a central regulator in the immune system. Its importance is underlined by its broad use as a therapeutic agent against various cancers of the immune system, and IL-2 antagonists are used to prevent rejection of transplanted organs. Recently, we determined the crystal structure of the heterotrimeric ectodomains of the receptor for IL-2 (IL-2Rαβγc) in complex with IL-2 at 3.0-Å resolution. On the basis of these results, we propose a stepwise assembly pathway for the quaternary IL-2 signaling complex initiated by the formation of the binary complex consisting of IL-2 and IL-2Rα. Because IL-2Rα makes no contacts with IL-2Rβ or IL-2Rγc and only minor changes occur in the IL-2 structure in response to receptor binding, IL-2Rα appears to deliver IL-2 to the signaling complex and act as a regulator of signal transduction. Cooperativity in assembly of the quaternary complex arises from the extensive interfaces found within the fully assembled IL-2 signaling complex. Helix A of IL-2 wedges tightly between IL-2Rβ and IL-2Rγc to form a 3-way junction that provides a composite binding site for the final recruitment of IL-2Rγc. This work was performed in collaboration with K.A. Smith, Weill Medical College, New York, New York.

Publications

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.

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.

Cheng, H., Chong, Y., Hwang, I., Tavassoli, A., Zhang, Y., Wilson, I.A., Benkovic, S.J., Boger, D.L. Design, synthesis, and biological evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as potent inhibitors of GAR Tfase and the de novo purine biosynthetic pathway. Bioorg. Med. Chem. 13:3577, 2005.

Cheng, H., Hwang, I., Chong, Y., Tavassoli, A., Webb, M.E., Zhang, Y., Wilson, I.A., Benkovic, S.J., Boger, D.L. Synthesis and biological evaluation of N-{4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl}-L-glutamic acid as a potential inhibitor of GAR Tfase and the de novo purine biosynthetic pathway. Bioorg. Med. Chem. 13:3593, 2005.

Choe, J., Kelker, M.S., Wilson, I.A. Crystal structure of human Toll-like receptor 3 (TLR3) ectodomain. Science 309:581, 2005.

Chong, Y., Hwang, I., Tavassoli, A., Zhang, Y., Wilson, I.A., Benkovic, S.J., Boger, D.L. Synthesis and biological evaluation of α- and γ-carboxamide derivatives of 10-CF3CO-DDACTHF. Bioorg. Med. Chem .13:3587, 2005.

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.

Giabbai, B., Sidobre, S., Crispin, M.D., Sanchez-Ruiz, Y., Bachi, A., Kronenberg, M., Wilson, I.A., Degano, M. Crystal structure of mouse CD1d bound to the self ligand phosphatidylcholine: a molecular basis for NKT cell activation. J. Immunol. 175:977, 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., 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.

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.

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.

Moody, D.B., Zajonc, D.M., Wilson, I.A. Anatomy of CD1-lipid antigen complexes. Nat. Rev. Immunol. 5:387, 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.

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.

Stauber D.J., Debler, E.W., Horton, P.A., Smith, K.A., Wilson, I.A. Crystal structure of the interleukin-2 signaling complex: paradigm for a heterotrimeric cytokine receptor. Proc. Natl. Acad. Sci. U. S. A., in press.

Van Rhijn, I., Zajonc, D.M., Wilson, I.A., Moody, D.B. T-cell activation by lipopeptide antigens. Curr. Opin. Immunol. 17:222, 2005.

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.

Wilson, I.A., Stanfield, R.L. MHC restriction: slip-sliding away. Nat. Immunol. 6:434, 2005.

Wiseman, R.L., Johnson, S.M., Kelker, M.S., Foss, T., Wilson, I.A., Kelly, J.W. Kinetic stabilization of an oligomeric protein by a single ligand binding event. J. Am. Chem. Soc. 127:5540, 2005.

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.

Zajonc, D.M., Cantu, C. III, Mattner, J., Zhou, D., Savage, P.B., Bendelac, A., Wilson, I.A., Teyton, L. Structure and function of a potent agonist for the semi-invariant natural killer T cell receptor. Nat. Immunol. 6:810, 2005.

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.

Zhang, Y., Wang, L., Schultz, P.G., Wilson, I.A. Crystal structures of apo wild-type M jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine. Protein Sci. 14:1340, 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.

 

Ian A. Wilson, D.Phil.
Professor

Wilson Web Site