Beesam, S. | Search bioRxiv

bioRxiv2022

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

TCR signaling poses a mechanical problem: pMHC binding occurs at the TCR{beta} 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 TCR{beta} and CD3 have been proposed to contribute to this coupling, their kinetics and mechanical competence remain elusive. Here, we quantify TCR{beta}-CD3 ECD cis-interactions in a pseudo-cis configuration using two-dimensional binding and single-bond force spectroscopy, finding that TCR{beta}-CD3{gamma}{varepsilon} and TCR{beta}-CD3{delta}{varepsilon} interactions have low affinity and rapid kinetics, yet form catch bonds. Critically, concurrent engagement of CD3{gamma}{varepsilon} and CD3{delta}{varepsilon} produces high cis-cooperativity, yielding a stronger and longer-lasting CD3{gamma}{varepsilon}-TCR{beta}-CD3{delta}{varepsilon} 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 CD3{delta}{varepsilon} more force-responsive and susceptible to conformational change than CD3{gamma}{varepsilon}. 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.

immunology↗

bioRxiv2022

In situ cell-surface conformation of the TCR-CD3 signaling complex

T cells play a vital role in adaptive immune responses to infections, inflammation and cancer and are dysregulated in autoimmunity. Antigen recognition by T cells - a key step in adaptive immune responses - is performed by the T cell receptor (TCR)-CD3 complex. The extracellular molecular organization of the individual CD3 subunits (CD3{delta}{varepsilon} and CD3 {gamma}{varepsilon}) around the {beta}TCR is critical for T cell signaling. Here, we incorporated unnatural amino acid (UAA) photo-crosslinkers at specific mouse TCR, TCR{beta}, CD3{delta} and CD3{gamma} sites, based on previous mutagenesis, NMR spectroscopy and cryo-EM evidence, and crosslinking allowing us to identify nearby interacting CD3 or TCR subunits on the mammalian cell surface. Using this approach, we show that CD3{gamma} and CD3{varepsilon}, belonging to CD3{gamma}{varepsilon} heterodimer crosslinks to C{beta} FG loop and C{beta} G strand, respectively and CD3{delta} crosslinks to C{beta} CC loop and C DE loop. Together with computational docking, we identify that in in situ cell-surface conformation, the CD3 subunits exists in CD3{varepsilon}-CD3{gamma}-CD3{varepsilon}-CD3{delta} arrangement around the {beta} TCR. This unconventional technique, which uses the native mammalian cell surface microenvironment, includes the plasma membrane and excludes random, artificial crosslinks, captures a dynamic, biologically relevant, cell-surface conformation of the TCR-CD3 complex, which is compatible with the reported static cryo-EM structures overall CD3 subunits arrangement, but with key differences at the TCR-CD3 interface, which may be critical for experiments in T cell model systems.

immunology↗