The Skaggs Institute
for Chemical Biology

Scientific Report 2007

Crystallographic Studies of Immune Recognition and Therapeutic Targets

I.A. Wilson, C. Bell, R.M.F. Cardoso, P.J. Carney, S. Connolly, D. Ekiert, M.-A. Elsliger, S. Ferguson, B.W. Han, M. Hong, R. Kirchdoerfer, M.J. Jimenez-Dalmaroni, R. Pejchal, A. Schiefner, R.L. Stanfield, R.S. Stefanko, J. Stevens, J.A. Vanhnasy, S. Yoon, J. Xu, D.M. Zajonc

We focus on the structure and function of proteins involved in modulating the adaptive and innate immune responses. A better understanding of these proteins is critical for the development of vaccines and drugs to counter microorganisms such as influenza virus, HIV type 1 (HIV-1), and bacterial pathogens. Most recently, we are targeting important viral epitopes, alone and in complex with antiviral antibodies, and molecules involved in the innate immune response, such as Toll-like receptors (TLRs) and CD1.

Influenza Virus Glycoproteins

Infections with influenza viruses are usually fairly innocuous except in the very young and the elderly, because of the widespread immunity of most of the population to the common viral strains. However, strains such as the one in 1918 that caused more than 20 million deaths or the more recent one that causes bird flu are a constant reminder of the imminent threat of another pandemic. To better understand why some strains of influenza virus are more virulent than others, and to design improved vaccines and drugs to block current and future viral strains, we are studying the 2 viral surface proteins, hemagglutinin and neuraminidase, from different influenza viruses.

We recently determined crystal structures of the N1 neuraminidase from the 1918 virus alone and in complex with the antiviral drug zanamivir. We found that a significant ligand-induced structural change occurs in the tetrameric neuraminidase in a loop region near the active site of the molecule (Fig. 1). This conformational change results in the formation of an extra cavity in the binding site when no inhibitor is present. Studies are now under way to design inhibitors to target this cavity.

In addition to our previously determined crystal structure for the hemagglutinin of the 1918 virus, we are now working toward a structure for the pH-induced, fusion-active form of the hemagglutinin to probe the mechanism used by the virus to enter target cells. We are also crystallizing other proteins of the 1918 virus, such as nonstructural protein 1, matrix 1 protein, nucleoprotein, and the polymerase complex.

Fig. 1. Crystal structure of the neuraminidase tetramer of the 1918 influenza virus. The tetramer is shown in green, with 1 of the 4 subunits colored from blue to red along the chain from the N terminus to the C terminus.

Strain H5N1 influenza (bird flu) viruses are not readily transmitted among humans, likely because of differences in the receptor specificity of the viral hemagglutinin. To investigate structural changes critical for receptor switching from avian to human specificity, we are developing a baculovirus display platform that will enable us to study large libraries of mutant hemagglutinins. We will select hemagglutinin variants with altered receptor specificity by panning our library against immobilized glycans or human bronchial cell monolayers. The receptor specificity of the selected variants will be determined by using glycan arrays. Structural changes associated with receptor switching will be characterized by using x-ray crystallography. Our goal is to elucidate the structural mechanisms through which mutations that affect receptor specificity and recognition by hemagglutinin contribute to the transmissibility of influenza virus in humans. Our research on influenza viruses is carried out as part of a flu consortium funded by the National Institute of Allergy and Infectious Diseases.

HIV Vaccine Design and Viral Cell Entry

Since the beginning of the AIDS epidemic in the early 1980s, more than 65 million persons have been infected with HIV-1, and 25 million have died. Currently, about 40 million persons are infected with HIV-1; about two-thirds of these live in sub-Saharan Africa. Although current drug treatments are effective at slowing the course of the infection, they do not eradicate the virus and are very expensive. Thus, a vaccine to prevent initial infection by HIV-1 is of extreme importance for slowing further spread of this virus. To this end, we are studying neutralization of HIV-1 by antibodies.

Only a few antibodies that effectively neutralize a wide range of HIV-1 isolates have been discovered, and we have determined structures of most of these in complex with the appropriate viral antigens. Using this structural information about the critical viral epitopes, we are trying to design antigens that can elicit a broad neutralization response. Recent advances include the crystal structure for Fab F425-B4e8 in complex with a peptide corresponding to the V3 region of the viral envelope protein gp120. Although most antibodies that target the V3 region are specific for a single viral isolate, F425-B4e8 is a cross-reactive anti-V3 antibody, one of several such antibodies discovered thus far.

The structure of the V3 peptide bound to F425-B4e8 is unusual; the peptide has a 5-residue α-turn around the V3 crown residues GPGRA (Fig. 2) rather than the more common 4-residue β-turn detected in previous V3 structures. The structure also shows that F425-B4e8 recognizes V3 primarily through interactions with just 2 side chains, isoleucine at position 309 and arginine at position 315. This recognition of a limited number of side chains likely adds to the enhanced cross-reactivity of this antibody.

We are also working to determine the structure of the viral gp140 protein in its native, trimeric form. Although both gp120 and gp140 associate as trimers on the viral surface, they are always found as monomers in solution, and the only structural data currently available are for the monomeric forms. The structure of a native gp140 trimer would be valuable for vaccine design, because this form of the protein is the form that neutralizing antibodies must recognize to effectively prevent viral infection.

In other research on HIV-1, our goal is to gain a better understanding of HIV-1 cell entry. Initially, the virus binds to a receptor (CD4) and then to a coreceptor (the chemokine receptors CCR5 or CXCR4) that is a member of the G protein–coupled receptor (GPCR) family. The GPCRs are a large family of integral membrane proteins that are central to numerous biological processes, including vision, smell, behavior and mood regulation, autonomic nervous system transmission, and regulation of the immune system. Together, the GPCRs constitute one of the largest known protein families. Their diversity of function is matched only by the exceptional variety of ligands they recognize. A corollary of this extensive involvement in normal biological processes is the role of GPCR signaling in many pathologic conditions.

Fig. 2. Fab F425-B4e8 bound to V3 peptide. The bound peptide is shown in yellow, with side chains labeled. This V3 peptide has an unusual α-turn around the GPGRA rather than a β-turn as previously noted for these peptides bound to anti-V3 antibodies.

In the immune system, the chemokine receptors and their chemokine ligands are central to intercellular communication and cell recruitment during inflammation. Furthermore, their involvement in disease progression is highlighted by the ability of several chemokine receptors to serve as coreceptors in the membrane-fusion mechanism of HIV. Because of their involvement in many diseases, GPCRs collectively account for more than 50% of current chemotherapeutic targets. In line with the involvement of chemokine receptors in immunomodulation and HIV infection, recently we have focused on developing recombinant expression systems for numerous chemokine receptors. Although yield and protein stability continue to be problems in the structural investigation of these proteins, the early results in protein production and solubilization with multiple methods appear promising.

Our studies on HIV work are done in collaboration with D.R. Burton and P.E. Dawson, Scripps Research; C.-H. Wong, Skaggs Institute; L.A. Cavacini, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; J.K. Scott, Simon Fraser University, Burnaby, British Columbia; J. Moore, Weill Medical College of Cornell University, New York, New York; H. Katinger, R. Kunert, and G. Stiegler, University of Agriculture, Vienna, Austria; R. Wyatt and P. Kwong, Vaccine Research Center, National Institutes of Health, Bethesda, Maryland; W. Olson and K. Kang, Progenics Pharmaceuticals, Inc., Tarrytown, NY; the National Institutes of Health; and the Neutralizing Antibody Consortium of the International AIDS Vaccine Initiative.

The Innate Immune Response Against Microbial Pathogens

CD1 molecules are nonclassical MHC class I antigen-presenting molecules. They present lipid, glycolipid, or lipopeptides for immune recognition. The CD1-lipid complexes are recognized by specific T cells through the cells' T-cell receptors (TCRs). During the past year, we have intensified our efforts to crystallize CD1-glycolipid-TCR complexes. To increase our success rate, we are examining a selection of 6 different human CD1-lipid combinations–CD1a-DDM838, CD1b–mycolic acid, CD1b-GMM, CD1c-MPD, CD1c-TritonX114, and CD1d-PPBF–that are recognized by the variant TCRs CD8.2, DN1, LDN5, CD8.1, YE2.3, and IL1ap, respectively. The cDNAs for the CD1 proteins and the TCRs, as well as the ligands, are provided through collaborations with M. Brenner and B. Moody, Harvard Medical School, Boston, Massachusetts. We have expressed CD1a, CD1d, and all 6 TCRs by using the baculovirus expression system, and we have developed a general purification protocol with TCRs CD8.2 and IL1ap. The CD1-lipid-TCR combinations are being evaluated for crystallization.

TLRs are human cell-surface receptors that detect microbes once the organisms have breached physical barriers such as the skin or intestinal tract mucosa; these receptors play a key role in initiating immune responses. TLRs recognize a variety of pathogen-associated molecular patterns, including components of bacterial cell walls and viral nucleic acids. Bacterial lipopolysaccharide, which is responsible for sepsis in humans, is recognized by TLR4 in complex with myeloid differentiation protein-2 (MD-2). After binding of lipopolysaccharide, TLR4 recruits intracellular adaptor molecules and initiates signaling. Currently, it is unclear how lipopolysaccharide recognizes TLR4 and activates TLR4 signaling. Structural studies on the formation of the complex consisting of TLR4, MD-2, and lipopolysaccharide are critical in providing insights into lipopolysaccharide specificity to TLR4 and lipopolysaccharide-induced TLR4 activation.

We expressed and purified the extracellular domain of TLR4 complexed with MD-2. Crystallization trials are under way to determine the crystal structure of the TLR4–MD-2–lipopolysaccharide complex. The results of this study will be useful in designing new TLR4 antagonists that target sepsis. This research is done in collaboration with B. Beutler and R. Ulevitch, Scripps Research.

Publications

Cardoso, R.M., Brunel, F.M., Ferguson, S., Zwick, M., Burton, D.R., Dawson, P.E., Wilson, I.A. Structural basis of enhanced binding of extended and helically constrained peptide epitopes of the broadly neutralizing HIV-1 antibody 4E10. J. Mol. Biol. 365:1533, 2007.

Cooper, Z.D., Narasimhan, D., Sunahara, R.K., Mierzejewski, P., Jutkiewicz, E.M., Larsen, N.A., Wilson, I.A., Landry, D.W., Woods, J.H. Rapid and robust protection against cocaine-induced lethality in rats by the bacterial cocaine esterase. Mol. Pharmacol. 70:1885, 2006.

Debler, E.W., Kaufmann, G.F., Kirchdoerfer, R.N., Mee, J.M., Janda, K.D., Wilson, I.A. Crystal structures of a quorum-quenching antibody. J. Mol. Biol. 368:1392, 2007.

Law, M., Cardoso, R.M., Wilson, I.A., Burton, D.R. Antigenic and immunogenic study of membrane-proximal external region-grafted gp120 antigens by a DNA prime-protein boost immunization strategy. J. Virol. 81:4272, 2007.

Lazoura, E., Lodding, J., Farrugia, W., Ramsland, P.A., Stevens, J., Wilson, I.A., Pietersz, G.A., Apostolopoulos, V. Enhanced major histocompatibility complex class I binding and immune responses through anchor modification of the non-canonical tumour-associated mucin 1-8 peptide. Immunology 119:306, 2006.

Muller, R., Debler, E.W., Steinmann, M., Seebeck, F.P., Wilson, I.A., Hilvert, D. Bifunctional catalysis of proton transfer at an antibody active site. J. Am. Chem. Soc. 129:460, 2007.

Nelson, J.D., Brunel, F.M., Jensen, R., Crooks, E.T., Cardoso, R.M., Wang, M., Hessell, A., Wilson, I.A., Binley, J.M., Dawson, P.E., Burton, D.R., Zwick, M.B. An affinity-enhanced neutralizing antibody against the membrane-proximal external region of human immunodeficiency virus type 1 gp41 recognizes an epitope between those of 2F5 and 4E10. J. Virol. 81:4033, 2007.

Sanguineti, S., Centeno Crowley, J.M., Lodeiro Merlo, M.F., Cerutti, M.L., Wilson, I.A., Goldbaum, F.A., Stanfield, R.L., de Prat-Gay, G. Specific recognition of a DNA immunogen by its elicited antibody. J. Mol. Biol. 370:183, 2007.

Saphire, E.O., Montero, M., Menendez, A., van Houten, N.E., Irving, M.B., Pantophlet, R., Zwick, M.B., Parren, P.W., Burton, D.R., Scott, J.K., Wilson, I.A. Structure of a high-affinity mimotope peptide bound to HIV-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies. J. Mol. Biol. 369:696, 2007.

Stanfield, R.L., Dooley, H., Verdino, P., Flajnik, M.F., Wilson, I.A. Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit binding. J. Mol. Biol. 367:358, 2007.

Stevens, ., Blixt, O., Paulson, J.C., Wilson, I.A. Glycan microarray technologies: tools to survey host specificity of influenza viruses. Nat. Rev. Microbiol. 4:857, 2006.

Stoll, R., Lee, B.M., Debler, E.W., Laity, J.H., Wilson, I.A., Dyson, H.J., Wright, P.E. Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA. J. Mol. Biol. 372:1227, 2007.

Tian, F., Debler, E.W., Millar, D.P., Deniz, A.A., Wilson, I.A., Schultz, P.G. The effects of antibodies on stilbene excited-state energetics. Angew. Chem. Int. Ed. 45:7763, 2006.

Xu, L., Chong, Y., Hwang, I., D'Onofrio, A., Amore, K., Beardsley, G.P., Li, C., Olson, A.J., Boger, D.L., Wilson, I.A. Structure-based design, synthesis, evaluation and crystal structures of transition state analogue inhibitors of inosine monophosphate cyclohydrolase. J. Biol. Chem. 282:13033, 2007.

Zajonc, D.M., Ainge, G.D., Painter, G.F., Severn, W.B., Wilson, I.A. Structural characterization of mycobacterial phosphatidylinositol mannoside binding to mouse CD1d. J. Immunol. 177:4577, 2006.