The DETCR is a surface membrane complex consisting of the ligand-binding clonotypic DE heterodimer in non- covalent association with the dimeric CD3 signaling subunits (CD3HJ, CD3HG and CD3]]) whose sensitivity and specificity are critical for protective host immunity. DE TCRs have been shown to manifest an unusual, dynamic and force-responsive mechanical regulation of pMHC ligand binding. Moreover, force induces different receptor conformers associated with energized and non-energized TCRDE ectodomain states. To focus on how information on TCR ligand occupancy is transduced into the cell, we have begun to study the TCRD transmembrane and cytoplasmic domain (TMC) structure. Unexpectedly, we observed that this domain forms a bipartite helix segmented by a dynamic hinge. The first transmembrane (TM) helix contains its two charged residues, R251 and K256, pointing outward from opposite sides with the latter controlling TM depth and all CD3 dimer associations. Mechanotransduction-mediated triggering of early T cell activation is modulated by these residues and, conversely, their disposition may be altered when physical force load is applied to the TCRDE- pMHC bond. Thus, rather than a robust static set of TM interactions, this DETCR dissociative mechanism implies the presence of dynamic structural conversions between and within segments. Here we shall exploit the latest advances in NMR direct detection techniques and relaxation and chemical exchange (Rex, CEST, etc.) along with EPR to directly observe states of individual and assembled components in membrane mimetics, their distributions and structures. The biological relevance of these structural observations shall be assessed through mutagenesis that restricts subunits to defined conformations or abrogates intersubunit interactions. Modified subunits that are assembled into cell surface DETCR complexes will be assessed for impact on pMHC-stimulated Ca2+ flux, IL-2 production and transcriptomes in T cells.
In Aim 1, we shall measure the dynamic behavior of the TCRDTMC alone or in complex with TCRE, CD3] and CD3G TM segments. TCRDTMC variants whose hinge is modified to lock in a bent or straightened configuration shall be compared along with that of pTD whose structure, predicted to be a straightened continuous helix responsible for high basal level activation in early DN3 thymocytes, shall be determined. The potential for TCRDTM K256 to be post-translationally modified during mechanotransduction by a methytransferase or other enzyme localized to the inner plasma membrane leaflet shall be assessed.
In Aim 2, similar studies will be performed with TCRE and CD3G alone or associated with other DETCR complex TM components. With respect to CD3G, structural and dynamic changes in both TM and cytoplasmic tail consequent to ITAM Tyr phosphorylation shall be defined. Given that the majority of medicinal compounds bind transmembrane segments of proteins, information gleaned herein shall be both of practical and fundamental significance to target resting or activated conformations.
The TCR mediates antigen-specific T cell stimulation important in protection against infections and cancers as well as autoimmune diseases. Insight from this proposal will contribute to TCR knowledge, with relevance for discovery of small molecule drugs and optimal adoptive T cell immunotherapies to control T cell activation.
