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

Initial molecular details of cellular activation following αβT-cell receptor (TCR) ligation by pMHC remain unexplored. We determined the NMR structure of the TCRα 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 CD3ζζ and CD3εγ/CD3εδ, respectively, leading to an activated transcriptome. Optical tweezers reveal that Arg251 and Lys256 mutations alter αβTCR-pMHC bond lifetimes, while mutations within interacting TCRα connecting peptide and CD3δ 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 TCRαβ and CD3 associations. This αβTCR dissociative mechanism impacts future immunotherapy and synthetic receptor design on CAR-T cells. Transcriptional profiling of wild type, R251L, K256L, and G259L/N261A T-cell lines