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.