During the last funding interval as a merit award, this grant focused on the T cell receptor (TCR) complex composed of an ?? heterodimer and non-covalently associated CD3 signaling components. We continued crystallographic analysis with TCRs, providing the first structure of an ?? heterodimer in complex with a peptide/MHC (pMHC) class II ligand. In conjunction with a crystallographic structure of CD4 D1D2 binding to the same MHC class II molecule, a hydrophobic concavity formed by residues from membrane proximal ?2 and ?2 MHC class II domains was revealed. We excluded a direct TCRab-CD4 interaction, instead revealing how TCR?? and CD4 signaling is coordinated in a """"""""V-shape"""""""" around the antigenic peptide/pMHC class II complex. Solution structures of CD3?? and CD3?? ectodomain complexes were determined by NMR, uncovering for each dimer a unique side-to-side hydrophobic interface between their two Ig-like domains with parallel pairing of respective C-terminal ?-strands, and suggesting how rigidified CD3 elements participate in TCR-based signal transduction. To now characterize the structural basis for early signal transduction events via this TCR?? complex, four aims are proposed involving the CD3?? heterodimer and associated TCR ? chain. First, NMR-based methods will be used to determine the precise binding site on CD3?? of activating and non-activating anti-CD3? mAbs, given equivalent binding affinities. Second, CD3??-associated TCR? chain recognition function during thymic development and T cell activation accounting for V? repertoire bias will be assessed. Third, the structure and function of the transmembrane (TM) segments of CD3? and ? will be determined and their interaction with that of TCR ? ascertained. Fourth, electron paramagnetic resonance (EPR) methods will be exploited to define the orientation and disposition of TM elements and how pMHC or anti-CD3? mAbs affect TM depth, orientation and structural conformation. Distance measurements between the ? chain and CD3? and CD3? ectodomains will also be examined before and after pMHC or anti- CD3 mAb ligation. That orthological rather than vertical force via CD3 components activates T cells suggests a dynamic mechanosensor TCR model;pMHC pulls on the TCR from the opposing antigen-presenting cell surface such that the ?? heterodimer then presses on the CD3 ectodomains to initiate signaling.
The TCR is responsible for mediating antigen-specific T cell stimulation. As such, insights from this proposal will be relevant for design of inhibitory and activating anti-TCR monoclonal antibodies and other molecules to treat human autoimmune and immunodeficiency states, respectively.
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