Molecular Interactions Of Lymphoid Cell Receptors

National Institute Of Allergy And Infectious Diseases

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Abstract

These projects take advantage of the laboratory's expertise in studying molecular interactions and molecular structure. For project (1) indicated above, we have developed a strategy for generating disulfide-trapped peptide/MHC-I molecules and have expressed, refolded, purified, and crystallized several of these. In particular, we have examined H2-Dd molecules in which a residue in the binding cleft has been mutated to cysteine and have refolded these with peptides containing cysteine in appropriate positions. X-ray crystallographic structures of these confirm that the disulfide-trapped peptides are stably fixed in the peptide binding groove. Versions of these, produced with truncated peptides, indicate that these molecules are peptide-stabilized in part of the peptide binding groove but are relaxed in the positions of truncation. Binding studies indicate that these molecules should allow mapping of sites of interaction with various cellular and viral chaperones. Using a slightly different approach, we have produced several variations of MHC-I molecules with novel intrachain disulfide bonds, specifically H2-Dd and H2-Ld. Part (2) of this project is the detailed characterization of these novel MHC molecules with the goal of extending the approach to improved ways of imaging cells that express surface ligands for MHC-I, namely CD8 T lymphocytes and NK cells. Further studies to investigate not only the stability, but the biological activity and utility as tetramer staining reagents are underway. In addition, we have taken advantage of several strategies for generating peptide/MHC complexes that covalently link peptide variants to MHC molecules, exploiting a disulfide trap approach. These molecules include the human HLA-B*44:05 molecule linked to various truncated peptides, as well as the human HLA-B*27:05 and HLA-B*27:09 molecules.