Structural Immunology

Research in our laboratory is directed at understanding the molecular basis of ligand recognition by cell surface receptors of the immune system. Four classes of recognition molecules are under study: antibodies, T cell receptors (TCRs), natural killer (NK) cell receptors, and peptidoglycan recognition proteins (PGRPs). In addition, we are examining the assembly and structure of T cell signaling complexes.

Structure-function studies of antigen-antibody reactions

Knowledge of the molecular basis of protein-protein recognition is essential for understanding protein function, since the ability of proteins to form specific complexes with other proteins underlies most cellular processes. We are using the anti-lysozyme antibodies D1.3 and HyHEL-63 as models for elucidating the principles governing protein-protein recognition. We have chosen to work with the protein-engineered, bacterially-expressed Fv fragments of D1.3 and HyHEL-63 since the three-dimensional structures of the free Fvs and of the Fv-lysozyme complexes are known to high resolution. Site-directed mutants designed to investigate particular aspects of the association reactions are being characterized by X-ray crystallography to precisely ascertain the effects of particular amino acid substitutions at the structural level. In parallel, we are using surface plasmon resonance and titration calorimetry to determine the affinity and entropy and enthalpy changes of the binding reactions with the aim of correlating these thermodynamic parameters with the X-ray models.

Physical basis of T cell activation by superantigens
Superantigens (SAGs) are proteins of bacterial or viral origin that stimulate T cells by cross-linking TCRs and major histocompatibility (MHC) class II molecules. Stimulation leads to hyperactive responses associated with the massive release of pyrogenic and inflammatory cytokines, usually followed by T cell anergy or deletion. Two classes of SAGs have been identified: exogenous soluble proteins secreted by bacteria, such as the staphylococcal enterotoxins, and endogenous retroviral-encoded transmembrane proteins, such as the SAGs of mouse mammary tumor viruses. To elucidate the physical basis of T cell activation by microbial SAGs, we are pursuing a multi-disciplinary approach involving (i) molecular biology to generate recombinant forms of the relevant TCR, MHC, and SAG molecules, (ii) biophysical methods to measure the affinity of their interactions, and (iii) high resolution X-ray crystallography to determine their three-dimensional structures. The structures of representative TCR-SAG and SAG-MHC complexes are revealing the diverse strategies SAGs have evolved for cross-linking TCR and MHC molecules, while engineered SAG mutants with altered binding properties for TCR and MHC are being used to define the kinetic and affinity parameters governing T cell activation by these potent microbial toxins.

Molecular mimicry in multiple sclerosis
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) mediated by autoreactive T cells. The activation of resting T cells reactive with CNS antigens is believed to be caused by bacterial or viral peptides with structural similarity to immunodominant self-peptides of the myelin sheath, such as MBP 86-105 (molecular mimicry). We have determined the three-dimensional structure of the human MHC class II molecule HLA-DR2a, a marker for increased susceptibility to MS, in complex with the MBP 86-105 self-peptide. Using random combinatorial libraries, a number of viral peptides, including ones derived from human herpes virus-6, have been identified that efficiently activate T cell clones specific for HLA-DR2a/MBP 86-105. Crystal structures of cross-reactive viral peptides bound to HLA-DR2a will allow us to compare the antigenic recognition surfaces of different MHC/peptide ligands that activate the same TCR, thereby providing insight into how microbial peptides mimic the MBP self-peptide.

Structural basis of MHC class I recognition by NK receptors
NK cells are a fundamental component of the innate immune with the intrinsic ability to recognize and destroy virally-infected and tumor cells that are deficient in MHC class I expression. The lack of normal MHC class I expression is detected by a class of MHC class I allele-specific receptors on NK cells. Potential targets are protected from lysis if they express sufficient levels of the appropriate MHC class I molecule. We are using X-ray crystallography, coupled with mutagenesis and direct binding measurements, to establish the molecular basis of ligand recognition by key receptors known to regulate NK cell-mediated cytotoxicity, either positively or negatively. These include members of the Ly49 family of NK receptors and 2B4.

Structural basis for peptidoglycan recognition by the innate immune system
In addition to MHC class I recognition through NK receptors, the innate immune system recognizes conserved products of microbial metabolism that are unique to microorganisms and are not produced by the host (pathogen-associated molecular patterns, or PAMPs). Examples of PAMPs include lipopolysaccharide (LPS) of Gram-negative bacteria and peptidoglycan (PGN) of Gram-positive bacteria. PGRPs show distinct specificities for PGNs from different microbes, but the structural basis for differential recognition is unknown. We are focusing on human peptidoglycan proteins (PGRPs), which bind PGNs on the cell wall of bacterial pathogens. Recently, we determined the crystal structure of one member of the human PGRP family. This study is currently being extended to other PGRPs and their complexes with specific PGN ligands.

Assembly and structure of T cell signaling complexes
The transmembrane adaptor protein LAT is essential for T cell activation following TCR ligation. LAT is believed to direct the assembly a multiprotein signaling complex comprising the adaptor molecules Gads and SLP-76, and the enzyme phospholipase Cgamma1 (PLCgamma1). While rapid progress is being made towards the identification of LAT-associated proteins, and on understanding their role in lymphocyte development and function, much less is known about the biochemical properties and structure of LAT-based signaling complexes. We are studying the assembly and structure of the postulated LAT/Gads/SLP-76/PLCgamma1 complex using a multidisciplinary approach combining biophysical methods to measure thermodynamic binding parameters with X-ray crystallography to determine three-dimensional structures. The results will contribute to the development of structure-based therapies for the treatment of T cell-mediated autoimmune diseases based on the specific disruption of LAT-nucleated signaling complexes by small molecule inhibitors of protein-protein association.