Our studies on HIV are done in collaboration
with D.R. Burton, M. Zwick, R. Pantophlet, P.E. Dawson, and C.-H. Wong, Scripps
Research; B. Davis, University of Oxford; L. Cavacini and J.K. Scott, Simon Fraser
University, Burnaby, British Columbia; J. Moore, Weill Medical College of Cornell
University, New York, New York;
Influenza Virus Glycoproteins
The 1918 influenza pandemic, which was responsible for more than 20 million deaths worldwide, and the more recent "bird flu," with its even higher mortality rate (about 60% of patients in whom it is diagnosed), are constant reminders of the potential devastation that could ensue if a new influenza pandemic were to occur. To aid in the design of a vaccine to protect against such highly virulent strains of influenza, we are carrying out structural and functional studies of envelope proteins of influenza virus in complex with neutralizing antibodies to the virus.
All known antibodies that neutralize influenza virus recognize the hemagglutinin viral envelope protein. In general, antibodies to hemagglutinin generally recognize highly variable epitopes at the membrane distal end of the hemagglutinin trimer. However, a small proportion of the host repertoire of antibodies is directed against other sites on hemagglutinin, including several antibodies that bind on the side of the hemagglutinin trimer and recognize highly invariant epitopes.
Several of these unusual antibodies neutralize the hemagglutinin of different strains and subtypes of influenza virus, both in vivo and in vitro. To gain insight into the mechanism of virus neutralization and the nature of the epitopes recognized, we are investigating several of these broadly neutralizing antibodies in complex with hemagglutinins that represent different pandemic strains and subtypes (H1, H2, H3) of human influenza virus as well as avian H5N1 influenza viruses. Understanding how these more broadly neutralizing antibodies interfere with viral entry and subsequent infection, as well as the nature of the highly conserved epitopes, will provide insights into the functional and antigenic constraints on the hemagglutinin of influenza virus.
Currently, we are working with 2 Fabs, H5M11 and H5M9, that neutralize the H5N1 avian influenza virus A/Goose/Guangdong/1/96 and more recent avian strains that have infected humans and with antibodies to the influenza virus that were isolated from survivors of the 1918 pandemic by J. Crowe, Vanderbilt University, Nashville, Tennessee. The structure of Fab H5M11 has been determined, and we are working toward crystallization of other Fabs in complex with hemagglutinins from the H5N1 and 1918 H1N1 viruses.
Hemagglutinin facilitates cell fusion through interactions with host membranes. Although crystal structures of hemagglutinin ectodomains have been extensively studied, little is known about the conformation or function of the membrane-interacting regions. We are working toward the determination of crystal structures of the full-length hemagglutinin in its states before and after fusion. In collaborative research with G. Tobin, Biological Mimetics, Inc., Frederick, Maryland, crystallization of the full-length hemagglutinin from A/Wyoming/3/03 (H3 subtype) and bacterial expression of the postfusion form of the protein are under way. We also propose to isolate and structurally analyze the hemagglutinin from the pandemic A/Japan/305/57 (H2 subtype) virus. These studies will advance our understanding of the mechanism of hemagglutinin-induced fusion and provide novel targets for design of fusion inhibitors.
The crystal structure of the neuraminidase of the 1918 H1N1 virus has been determined to 1.45 Å. A large cavity in the active site in the neuraminidase offers new opportunities for structure-based drug design. Crystal structures of the 1918 neuraminidase in complex with the antiviral drugs oseltamivir (Tamiflu) and zanamivir (Relenza) show that the loop bordering the cavity is extremely flexible in binding substrates, a characteristic that may indicate that the 1918 neuraminidase can bind more chemically diverse ligands than can neuraminidases from some other subtypes of the virus. This high-resolution structural information is being used for rational design of inhibitors against influenza virus.
Additional collaborators in the influenza research include our colleagues in the flu consortium funded by the National Institute of Allergy and Infectious Diseases; scientists at Crucell, Leiden, the Netherlands; J. Crowe, Vanderbilt University; A. Lanzavecchia, Institute for Research in Biomedicine, Bellinzona, Switzerland; and X. Che, Southern Medical University China, Guangzhou, China.
The Innate Immune Response Against Microbial Pathogens
Toll-like receptors (TLRs) are glycoproteins that are essential for innate immune recognition of microbial pathogens. The TLR extracellular domains are horseshoe-shaped molecules consisting of leucine-rich repeat domains that begin and end with an N- and a C-terminal cap domain. Recently, we have been studying the TLR4, which plays an essential role in recognition and signaling of bacterial lipopolysaccharide. Among the TLR family members, TLR4 is unique in requiring another molecule, myeloid differentiation protein-2 (MD-2), for its function. MD-2 directly binds lipopolysaccharide and induces dimerization and activation of TLR4 for signaling.
To provide insights into the structural mechanism used by lipopolysaccharide to activate TLR4, we have expressed the extracellular domain of TLR4 (sTLR4) in complex with MD-2 in a baculovirus expression system. Biophysical studies suggest that purified 1:1 complexes of sTLR4–MD-2 homodimerize to form 2:2 complexes in the presence of lipopolysaccharide. X-ray crystallographic studies are being carried out to reveal the structural architecture of the sTLR4–MD-2 assembly induced by lipopolysaccharide.
Jawless fish, such as the lamprey, do
not have immune receptors, such as antibodies, T-cell receptors, or MHC molecules,
yet the fish still have an adaptive immune response to antigen. Recently, it was
shown that cell-surface molecules, termed variable lymphocyte receptors (VLRs) are
responsible for the adaptive immune response in jawless fish. These receptors resemble
the mammalian innate system TLRs, with an overall horseshoe shape made up of a variable
number of different leucine-rich repeat domains. In collaboration with M. Cooper,
Emory University, Atlanta, Georgia, we recently determined the first crystal
structure of a VLR-antigen complex, RBC36, in complex with the H trisaccharide derived
from the H antigen of human type O erythrocytes (Fig. 2). This structure reveals
for the first time the location and nature of the VLR antigen-binding site.
The CD1 family of innate receptors consists
of MHC class I–like, antigen-presenting molecules that present lipids, glycolipids,
and lipopeptides to effector T cells. The receptors are expressed on antigen-presenting
cells and are involved in host defense and in immunoregulatory functions. Glycolipids
presented by CD1d are capable of stimulating natural killer T cells. Natural killer
Astronomo, R.D., Lee, H.K., Scanlan, C.N., Pantophlet, R., Huang, C.Y., Wilson, I.A., Blixt, O., Dwek, R.A., Wong, C.H., Burton, D.R. A glycoconjugate antigen based on the recognition motif of a broadly neutralizing human immunodeficiency virus antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the self glycans of gp120. J. Virol. 82:6359, 2008.
Bell, C.H., Pantophlet, R., Schiefner, A., Cavacini, L.A., Stanfield, R.L., Burton, D.R., Wilson, I.A. Structure of antibody F425-B4e8 in complex with a V3 peptide reveals a new binding mode for HIV-1 neutralization. J. Mol. Biol. 375:969, 2008.
Burton, D.R., Wilson, I.A. Immunology: square-dancing antibodies. Science 317:1507, 2007.
Debler, E.W., Kaufmann, G.F., Meijler, M.M., Heine, A., Mee, J.M., Pljevaljcic, G., Di Bilio, A.J., Schultz, P.G., Millar, D.P., Janda, K.D., Wilson, I.A., Gray, H.B., Lerner, R.A. Deeply inverted electron-hole recombination in a luminescent antibody-stilbene complex. Science 319:1232, 2008.
Debler, E.W., Müller, R., Hilvert, D., Wilson, I.A. Conformational isomerism can limit antibody catalysis. J. Biol. Chem. 283:16554, 2008.
Demartino, J.K., Hwang, I., Connelly, S., Wilson, I.A., Boger, D.L. Asymmetric synthesis of inhibitors of glycinamide ribonucleotide transformylase. J. Med. Chem. 51:5441, 2008.
Dhillon, A.K., Stanfield, R.L., Gorny, M.K., Williams, C., Zolla-Pazner, S., Wilson, I.A. Structure determination of an anti-HIV-1 Fab 447-52D-peptide complex from an epitaxially twinned data set. Acta Crystallogr. D Biol. Crystallogr. 64:792, 2008.
Huang, C.C., Lam, S.N., Acharya, P., Tang, M., Xiang, S.H., Hussan, S.S., Stanfield, R.L., Robinson, J., Sodroski, J., Wilson, I.A., Wyatt, R., Bewley, C.A., Kwong, P.D. Structures of the CCR5 N terminus and of a tyrosine-sulfated antibody with HIV-1 gp120 and CD4. Science 317:1930, 2007.
Johnson, S.M., Connelly, S., Wilson, I.A., Kelly, J.W. Biochemical and structural evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors. J. Med. Chem. 51:260, 2008.
Menendez, A., Calarese, D.A., Stanfield, R.L., Chow, K.C., Scanlan, C.N., Kunert, R., Katinger, H., Burton, D.R., Wilson, I.A., Scott, J.K. A peptide inhibitor of HIV-1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120. FASEB J. 22:1380, 2008.
Relloso, M., Cheng, T.Y., Im, J.S., Parisini, E., Roura-Mir, C., DeBono, C., Zajonc, D.M., Murga, L.F., Ondrechen, M.J., Wilson, I.A., Porcelli, S.A., Moody, D.B. pH-dependent interdomain tethers of CD1b regulate its antigen capture. Immunity 28:774, 2008.
Stevens, J., Blixt, O., Chen, L.M., Donis, R.O., Paulson, J.C., Wilson, I.A. Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity. J. Mol. Biol. 381:1382, 2008.
Verdino, P., Aldag, C., Hilvert, D., Wilson, I.A. Closely related antibody receptors exploit fundamentally different strategies for steroid recognition. Proc. Natl. Acad. Sci. U. S. A. 105:11725, 2008.
Wei, C.J., Xu, L., Kong, W.P., Shi, W., Canis, K., Stevens, J., Yang, Z.Y., Dell, A., Haslam, S.M., Wilson, I.A., Nabel, G.J. Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza virus. J. Virol. 82:6200, 2008.
Zajonc, D.M., Savage, P.B., Bendelac, A., Wilson, I.A., Teyton, L. Crystal structures of mouse CD1d-iGb3 complex and its cognate Vα14 T cell receptor suggest a model for dual recognition of foreign and self glycolipids. J. Mol. Biol. 377:1104, 2008.
Zajonc, D.M., Wilson, I.A. Architecture of CD1 proteins. Curr. Top. Microbiol. Immunol. 314:27, 2007.
Zhu, X., Xu, X., Wilson, I.A. Structure determination of the 1918 H1N1 neuraminidase from a crystal with lattice-translocation defects. Acta Crystallogr. D Biol. Crystallogr. 64:843, 2008.