Cooperative ectodomain interaction among TCRαβ, CD3γε, and CD3δε enhances TCR mechanotransduction

ABSTRACT TCR signaling poses a mechanical problem: pMHC binding occurs at the TCRαβ ectodomain (ECD) head, whereas ITAM phosphorylation occurs on CD3 cytoplasmic tails. Chemistry cannot bridge this >10 nm gap, requiring the two events to be coupled through the TCR–CD3 interface, thus involving conformational allostery and being regulatable by force. Although weak ECD cis -interactions between TCRαβ and CD3 have been proposed to contribute to this coupling, their kinetics and mechanical competence remain elusive. Here, we quantify TCRαβ–CD3 ECD cis -interactions in a pseudo- cis configuration using two-dimensional binding and single-bond force spectroscopy, finding that TCRαβ–CD3γε and TCRαβ–CD3δε interactions have low affinity and rapid kinetics, yet form catch bonds. Critically, concurrent engagement of CD3γε and CD3δε produces high cis -cooperativity, yielding a stronger and longer-lasting CD3γε–TCRαβ–CD3δε trimolecular catch bond than the sum of the two dimeric bonds, with force-stabilized lifetimes matching those of agonist TCR–pMHC trans -interaction. Molecular dynamics simulations reveal an expanded, cooperative, and asymmetric contact network, making CD3δε more force-responsive and susceptible to conformational change than CD3γε. Interface mutations do not alter force-free affinities but remodel cooperative cis -bond profiles, leading to an inverse correlation with trans -bond profiles and T cell signaling. These results identify cooperative ECD cis -interaction as a mechanically regulatable allosteric coupling element at the TCR–CD3 junction important to antigen recognition and signal initiation.