Fig. 2.
Identification of specific TCRαβ TM contacts within the receptor complex. (A) Summary of observed TCRαβ TM cross-links indicated by solid lines between cysteine-substituted positions. The F×E cross-link highlighted in the text is marked in red. Residue numbering throughout the paper starts at the CP cysteines responsible for the native intermolecular TCRαβ disulfide bond. (B) Panel of cross-link–positive combinations from the primary disulfide scan shown in Fig. S2. The indicated control (WT, cysteineless) or cysteine mutant human A6 TCRα (HA-tagged) and TCRβ [streptavidin-binding peptide (SBP)-tagged] mRNAs were cotranslated with a master mix of human CD3 and ζ mRNAs in in vitro assembly reactions and treated with 1 mM copper(II):phenanthroline (CuPhe) in TBS to induce TM disulfide bond formation. Digitonin-extracted products were immunoprecipitated using mAb OKT3 (anti-CD3δε/CD3γε) and separated by nonreducing SDS/PAGE. IP control panels demonstrate the specificity of product capture using WT and two of the strongest cross-link combinations; each indicated assembly reaction was split and subjected to an IP with specific (OKT3) or isotype-matched irrelevant control (Ctrl) antibodies. (C) Aliquots of the same reactions shown in B were subjected to CD3δ (PC-tagged) → CD3γ (FLAG-tagged) sequential nondenaturing IP (snIP) (19) to isolate minimally hexameric (CD3δε:TCRαβ:CD3γε) complexes and analyzed as above. The IP controls contained isotype-matched irrelevant (Ctrl) antibodies in both steps of the snIP procedure. Asterisks (*) mark the combinations counted as cross-link positive. (D) CuPhe dependence of TCRα-F26C × TCRβ-E20C (F×E) cross-linking and ζζ association. The indicated TCR combinations were processed as in B either with (+) or without (−) CuPhe addition after assembly and analyzed by snIP to select minimally hexameric complexes (Left) or only fully assembled ζζ-containing complexes (Right). Control (Ctrl) snIPs were performed as above on a duplicate CuPhe-treated WT assembly reaction, but used biotin-blocked streptavidin (SA) in lieu of an isotype control in the first step of TCRβ → ζ snIP. (E) Comparison of TM cross-linking in the presence and absence of the native CP-region disulfide bond. Assembled and CuPhe-treated complexes containing WT, F×E on the cysteineless background (F×ECS) or F×E on an otherwise WT background with CP disulfide bond intact (F×EWT) were captured from digitonin lysates using SA beads to bind the SBP-tagged TCRβ chain. The final wash step was performed with (+) or without (−) 10 mM TCEP in digitonin solution to selectively reduce EC but not TM disulfide bonds.