Parts (1) and (2) as listed above, deal with the MHC-I aspects of this project, and in general are directed to understand the molecular details of the loading of MHC-I molecules with self or antigenic peptides. Although hundreds of MHC-I and MHC-I-like three-dimensional structures have been determined, none of these is of a peptide-receptive (PR) form of the molecule. In previous studies, we determined the three-dimensional structure of a peptide epitope representative of a portion of the MHC-I molecule H2-Ld that is exposed only on partially unfolded, peptide receptive (PR) MHC-I molecules. This then defined one aspect of the PR form of the molecule, which was used as the input for molecular dynamics simulations of a fully hydrated model. This dynamics simulation has been examined extensively and provides a structural understanding of the way that MHC-I molecules change their shape from the metastable PR form to their stable PL form. Analysis of the two structures and of the transition from PR to PL suggests a detailed mechanism of how the MHC-I molecule works. To extend our understanding of the nature of peptide-loading, we have engineered the main chaperones involved in MHC-I loading, tapasin and Erp57. In addition, we have engineered a tapasin like molecule, known as TAPBPR, which is about 20% identical in amino acid sequence to tapasin and have undertaken studies examining the nature of its binding to the PR form of MHC-I. These studies suggest that TAPBPR interacts with a peptide-free, peptide-receptive form of MHC-I, and that this interaction is relaxed upon peptide binding. Additional studies of the TAPBPR/MHC-I interaction reveal direct interaction of antigenic peptides with the MHC-I and not the TAPBPR component of the complex. Furthermore, the interaction of peptide with the MHC-I molecule is quantitatively related to the strength of binding (the affinity) of the peptide for the MHC-I molecule. In additional studies of MHC-I/peptide interactions, we have explored the role that the anti-retroviral drug, abacavir, plays in binding to MHC-I and distorting the self-peptide repertoire bound by susceptible MHC-I alleles. In particular, we have shown, by peptide sequence analysis of the self peptides bound to the susceptible MHC-I allele, HLA-B*57:01, in the presence and absence of abacavir, that this drug can change the peptides that B*57:01 binds. This provides an explanation for the severe hypersensitivity reactions that are observed in a high proportion of HLA-B*57:01 individuals who receive the drug. We have developed transgenic mouse lines expressing various forms of HLA-B*57:01 as animal models for the effects of drugs in causing acute hypersensitivity reactions. These transgenic animals can be immunized to generate HLA-B*57:01-specific CD8-T cell responses. The third part of this project is focused on structural and functional studies of T cell receptor recognition of antigens, how this leads to T cell signaling, and how this leads to autoimmune disease. To provide a baseline for understanding antigen-specific structural changes in the TCR, we have determined the X-ray structure of a viral specific, MHC-I-restricted TCR, as well as its complex with its MHC-I/viral antigen ligand. Remarkably, although the MHC/peptide complex has a relatively rigid structure, the TCR shows great movement of its CDR3 alpha and beta loops, indicative of a fly-casting mechanism for ligand engagement. Further characterization of this fly-casting mechanism is reflected in other projects from this laboratory. Additional studies are underway to explore TCR/MHC-I interactions by novel biophysical techniques. In particular we have collaborated in studies that explore measurements of two-dimensional affinities of TCR/MHC interactions. In addition, we have collaboratively explored the use of NMR to study conformational changes in the TCR that accompany binding of a high affinity peptide/MHC complex. These studies offer new insight into possible allosteric mechanisms that contribute to T cell activation. Other approaches to understanding the TCR mediated aspects of autoimmunity include: 1) the characterization of antigenic peptides recognized by the autoimmune T cells in transgenic mouse models of autoimmune gastritis; and 2) the structural determination of MHC-II molecules bound to their antigenic peptides. We have determined the high resolution X-ray structure of IAd in complex with the Th2 peptide known as PLL, as well as the structures of two other IAd/peptide complexes in which the peptides are related to PLL, but are of higher intrinsic affinity. These structures, determined at high resolution reveal a previously unrecognized binding motif (exploiting residues 1,4,6,7, and 9 of the peptide) for IAd, particularly with respect to the preference of glutamic acid at position 9 of the peptide. This provides a framework for understanding interactions of autoimmune TCR with self MHC-II/peptide complexes. These experimental structural studies permitted the modeling of another gastritis-inducing peptide, known as PIT, bound to IAd, and provide further insight into the molecular basis of autoimmune gastritis. Similarities in this theme of the relevant anchors and the topology of the peptide bound to the MHC are consistent with other MHC-II/autoantigen complexes and suggest some common features that may be specifically relevant to autoimmune antigens.
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