During the next funding interval, we shall extend progress in areas of TCR-based recognition of pMHCI and II ligands and attendant co-receptor interactions to focus on the structural basis of TCR-proximal signaling events involving the CDS components. First, we will use information gained from our elucidation of the CDSey ectodomain fragment to design suitable CD3e8 constructs for structural work. Once structural information is obtained, then direct comparison of CD3ev and CDSeSheterodimers will be made. Their interaction with the TCRap recognition unit will be assessed by NMR using 15N-labeled CDSsy and CD3e8 subunits (whose interdomain linker has been removed) and unlabeled DIG and N15 TCRap proteins. Chemical shift analysis will identify which CDS residues contact or are conformalionally affected by TCRap.. Assessment of the CD3 binding sites on the TCRap molecules themselves can then be obtained by reciprocally J5N labeling either TCRoc or TCRp and assessing binding of unlabeled CDS dimers by NMR. Additional studies will involve crystallographic efforts to assemble TCR""""""""p, CDSey and CD3e8. Second, analysis of the CD3ey structure indicates that the CD3y Ig-like domain residues Q76, Y78 and Y79 and stalk residues C82 and C85 are likely involved in CDSey-dependent signaling. Both retroviral transduction of mutant CD3y using pLZRS-eGFP in CD3y/- FTOC and transgenesis in B6 and CD3ff- mice using tctracyline rcgulatable systems are being developed for analysis of the role of CD3y in pre-TCR and TCR functions in vivo. Similar analysis of CD38 mutants will be investigated once structural detail is;uncovered. Third, we will investigate structural rearrangements within the TCR complex upon pMHC binding using electron paramagnetic spectroscopy (EPS) to provide insight into the mechanism by which signal transduction is initiated. This approach will allow the investigation of dynamic events of large protein complexes that are otherwise inaccessible by crystallography or NMR. Suitable spin-labeled TCR complexes will be obtained based on the above structural information and EPS spectra monitored for changes in spectra line shape, allowing suitable movements such as rotational or piston-like movements to be examined in the TCR complex alone, upon addition of pMHCII (CA-I-Ak for D10) or an Fab fragment of the 3D3 anti-clonotypic mAb. The completion of these experiments will be instrumental in defining specific modes of movement and illuminating the structural mechanism by which TCR signaling is initiated.
Showing the most recent 10 out of 95 publications
