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 provided the first structure of an TCR 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 TCR -CD4 interaction, instead showing 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. Since tangential rather than vertical force via CD3 components activates T cells, a dynamic mechanosensor TCR model was proposed. In this 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. To now characterize the structural basis for early signal transduction events via the TCR complex, three aims will be pursued involving the CD3 heterodimers. First, NMR-based methods are proposed to determine the structure of individual CD3 , ? and transmembrane (TM) segments in isolation or as dimeric components using micelle, bicelle and nanodisc technologies. Functional consequences of mutations of residues involved in TM-TM interactions or other relevant structural features will be analyzed through gene knock-in or retroviral transduction into CD3 subunit null T cells. Second, the membrane proximal CxxC ectodomain motif present in each CD3 heterodimeric subunit will be structurally and functionally defined; its impact on individual TM structure will be determined under different redox states. As this disulfide-bonded structure caps the TM helix, reduction will presumably alter TM structure and signaling. Physiologic chemical or protein reductants of the CxxC motif will be assessed. Third, consequences of CxxC redox state on T cell activation, TCR complex quaternary structure topology and mechanosensor function will be determined. The CxxC segment likely serves as an attenuator of T cell activation in areas of ongoing inflammation.
The TCR is responsible for mediating antigen-specific T cell stimulation. As such, insights from this proposal will be relevant for design of small molecule as well as monoclonal antibody inhibitors and activators to treat human autoimmune and immunodeficiency states, respectively.
