In the first major aspect of this project, we have extended our understanding of the interactions of the NK cell activating receptor and T cell coreceptor, NKG2D, with its stress induced ligands. Although the structural details of the interaction of NKG2D with human MICA and ULBP3 are known, the interaction with the mouse RAE1 is only known at low resolution, and several other murine stress induced ligands of the same family have not been studied in detail. To this end, we engineered two of the previously less studied murine NKG2D ligands, MULT1 and H60, purified them, determined quantitatively their interactions with NKG2D by surface plasmon resonance, and determined several critical X-ray structures that permit us to understand the evolution of these molecules. We have determined the structure of MULT1 unliganded to 2.8 resolution, and a complex of H60 with NKG2D to a resolution of 3.3. These structures were then confirmed by the production of a battery of mutant MULT1 and H60 molecules and measurement of their affinities for NKG2D. These studies provide additional insight into the general problem of how NKG2D can recognize a large number of distinct ligands (8 different ligands in the human and 9 in the mouse) that are structurally related but are varied in amino acid sequence identity (ranging from 10 to 40%). The second sub-project here is a collaboration with the laboratory of John Coligan in which we have contributed our expertise in measurement of protein/protein interactions and have begun to address the question of the specificity of the interaction of the CD300 family of NK cell/ myeloid cell proteins with a variety of molecules founds on cell surface molecules that can be introduced into lipsomes. Data suggest that we can explore the interaction of CD300 family members with liposomes made with various compositions of phospholipids, an area that should prove valuable in understand the ligand specificity of these molecules. To add a structural basis to the biophysical description of the interaction of CD300 with phospholipids, we have recently crystallized and determined the structure of CD300f. The binding domain of this molecule has an Ig-like fold and we are actively exploring a structural understanding of the way such a molecule interacts with phospholipid ligands. Studies of interactions of such myeloid cell based receptors offer insight into a host of molecules involved in recognition of dead and dying cells based on interactions with cellular phospholipids. We have continued our interest in MHC/peptide interactions by determeining the X-ray structure of several variant peptides bound to the MHC molecule H-2Dd molecule to understand their role in immunization for CD8 T cell responses. In the last ongoing subproject, we are collaborating in efforts to engineer the cytokine IL-1beta and various mutants for targeting, via coupling to particular antibodies, to unique subsets of T lymphoctyes. We have examined the biological activities of recombinant IL-1beta and a set of its mutants, and are exploring various ways to couple the IL-1 to antibodies, by both biochemical or by genetic engineering approaches.
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