TCRa transmembrane domain coordinates antigen triggering by regulating bilayer immersion and CD3 associations

Initial molecular details of cellular activation following aßT-cell receptor (TCR) ligation by pMHC remain unexplored. We determined the NMR structure of the TCRa subunit transmembrane (TM) segment revealing a bipartite helix whose segmentation fostersdynamic movement. Positively charged TM residues Arg251 and Lys256 project from opposite faces of the helix, with Lys256 controlling immersion depth. Their modification causes step-wise reduction in associations with T-cell surface CD3?? and CD3e?/CD3ed, respectively, leading to an activated transcriptome. Optical tweezers reveal that Arg251 and Lys256 mutations alter aßTCR-pMHC bond lifetimes, while mutations within interacting TCRa connecting peptide and CD3d CxxC motif juxtamembrane elements selectively attenuate signal transduction. Our findings suggest that mechanical forces applied during pMHC ligation initiate T-cell activation by altering the disposition of those basic sidechains to rearrange TCR complex membrane topology and weaken TCRaß and CD3 associations. This aßTCR dissociative mechanism impacts future immunotherapy and synthetic receptor design on CAR-T cells. Overall design: Transcriptional profiling of wild type, R251L, K256L, and G259L/N261A T-cell lines