Part one, the MHC-I aspect of this project, is directed to understand the molecular details of the interaction between the peptide receptive (PR) form of MHC-I molecules and antigenic peptides. The description of this work was recently published (Mage, M., Dolan, M., Wang, R., Boyd, L., Revilleza, M.J., Robinson, H.J., Natarajan, K., Myers, N., Hansen, T., and Margulies, D.H.,The Journal of Immunology, 189:1291-1399 (2012), The peptide receptive transition state of MHC-I molecules: Insight from the structure of an alpha1 domain segment and molecular dynamics simulations). Although hundreds of MHC-I and MHC-I-like three-dimensional structures have been determined, none of these is of a PR form of the molecule. In these studies, we determined the three-dimensional structure of a peptide epitope representative of a portion of the MHC-I molecule H-2Ld that is exposed only on partially unfolded, peptide receptive (PR) MHC-I molecules. The determination of this structure was achieved by recognizing that a unique monoclonal antibody, 64-3-7, binds only to PR molecules and not to peptide loaded (PL) molecules. These observations, originally made in the laboratory of our collaborator, Dr. Ted Hansen, of Washington University at St. Louis, were further examined in our laboratory by an extensive binding analysis using surface plasmon resonance to ascertain the binding interaction of the 64-3-7 monoclonal antibody with various truncated peptides derived from the H-2Ld molecule. Then, following extensive efforts to obtain diffraction quality crystals of the Fab fragment of 64-3-7 with the full H-2Ld molecule, we successfully obtained crystals of the Fab protein complex with each of three different, but overlapping peptides. Synchrotron diffraction data were collected on four such crystals and all data sets were appropriately scaled. Molecular replacement solutions of the structure were obtained using homologous antibody fragments as the search model, and the structures were refined to high resolution (from 1.64 to 2.0 angstrom resolution), using several programs, including CNS, Refmac, and Phoenix. The four structures determined were essentially identical, and revealed that a portion of the 64-3-7 epitope (a sequence of seven amino acids) remains intact as a 3,10 helix in the Fab-complexed form, with several amino acid side chain adjustments. This suggests that this seven-residue peptide forms a molecular hinge that is first exposed to solvent in the PR form, and subsequently several of its side chains are then sequestered from solvent in the PL form. To visualize the structure of the entire peptide receptive, PR form of the H-2Ld molecule, the structure of the seven-residue epitope was spliced (in silico) into the known crystal structure of the complete H-2Ld molecule. To confirm the veracity of this model, and that the antibody 64-3-7 was capable of recognizing this same epitope as spliced into the context of the whole H-2Ld molecule, we performed a molecular docking simulation, using the Rosetta Dock program run on the high-performance computational resource of the Biowulf Linux cluster at the NIH. Of 10,000 docking solutions generated, the top scoring 10% were identified and of the 10 top scoring clusters, one solution containing a loop conformation with the residues of the peptide epitope in a similar conformation to those observed in the crystal structures was obtained. This then defined the PR form of the molecule, which was used as the input for molecular dynamics simulations of a fully hydrated model, using NAMD, a scalable molecular dynamics program, also run on the NIH Biowulf Cluster. 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. Thus, for the first time, we have dynamic images of the conformational changes that accompany the transition from peptide receptive to peptide bound forms of the MHC molecule. Detailed analysis of the two structures and of the transition from PR to PL suggest a detailed mechanism of how the MHC-I molecule works. The second part of this project is focused on structural and functional studies of T cell receptor recognition of autoantigens and how this leads to autoimmune disease. Our current approaches include: 1) the characterization of antigenic peptides recognized by the autoimmune T cells in transgenic mouse models of autoimmune gastritis;2) the structural determination of MHC-II molecules covalently linked to their antigenic peptides;and 3) the molecular characterization of MHC-II molecules loaded with autoimmunogenic peptides. Previous work from our laboratory showed that two different T cell receptors derived from mice with autoimmune gastritis were differentially pathogenic in inducing disease. One caused acute, flagrant disease and a Th1 response, one caused indolent disease and a Th2 response. We have determined the antigenic peptides recognized in the context of the MHC-II molecule, IAd, by these two TCR, and defined the frame of binding and the crucial binding and TCR residues of the peptides. In addition 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 2.5 angstrom resolution (and the others to somewhat lower 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.

Project Start
Project End
Budget Start
Budget End
Support Year
29
Fiscal Year
2012
Total Cost
$429,604
Indirect Cost
City
State
Country
Zip Code
Morozov, Giora I; Zhao, Huaying; Mage, Michael G et al. (2016) Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing. Proc Natl Acad Sci U S A 113:E1006-15
Liu, Baoyu; Chen, Wei; Natarajan, Kannan et al. (2015) The cellular environment regulates in situ kinetics of T-cell receptor interaction with peptide major histocompatibility complex. Eur J Immunol 45:2099-110
Margulies, David H (2014) The in-betweeners: MAIT cells join the innate-like lymphocytes gang. J Exp Med 211:1501-2
Sgourakis, Nikolaos G; Natarajan, Kannan; Ying, Jinfa et al. (2014) The structure of mouse cytomegalovirus m04 protein obtained from sparse NMR data reveals a conserved fold of the m02-m06 viral immune modulator family. Structure 22:1263-73
Murakami, Y; Tian, L; Voss, O H et al. (2014) CD300b regulates the phagocytosis of apoptotic cells via phosphatidylserine recognition. Cell Death Differ :
Mage, Michael G; Dolan, Michael A; Wang, Rui et al. (2013) A structural and molecular dynamics approach to understanding the peptide-receptive transition state of MHC-I molecules. Mol Immunol 55:123-5
Horai, Reiko; Silver, Phyllis B; Chen, Jun et al. (2013) Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen. J Autoimmun 44:21-33
Norcross, Michael A; Luo, Shen; Lu, Li et al. (2012) Abacavir induces loading of novel self-peptides into HLA-B*57: 01: an autoimmune model for HLA-associated drug hypersensitivity. AIDS 26:F21-9
Zhi, Li; Mans, Janet; Paskow, Michael J et al. (2010) Direct interaction of the mouse cytomegalovirus m152/gp40 immunoevasin with RAE-1 isoforms. Biochemistry 49:2443-53
Margulies, David H (2009) Home schooling of NK cells. Immunity 30:313-5

Showing the most recent 10 out of 13 publications