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

The extracellular molecular organization of the individual CD3 subunits around the αβ T cell receptor (TCR) is critical for initiating T cell signaling. In this study, we incorporate photo-crosslinkers at specific sites within the TCRα, TCRβ, CD3δ, and CD3γ subunits. Through crosslinking and docking, we identify a CD3ε'-CD3γ-CD3ε-CD3δ arrangement situated around the αβTCR in situ within the cell surface environment. We demonstrate the importance of cholesterol in maintaining the stability of the complex and that the 'in situ' complex structure mirrors the structure from 'detergent-purified' complexes. In addition, mutations aimed at stabilizing extracellular TCR-CD3 interfaces lead to poor signaling, suggesting that subunit fluidity is indispensable for signaling. Finally, employing photo-crosslinking and CD3 tetramer assays, we show that the TCR-CD3 complex undergoes minimal subunit movements or reorientations upon interaction with activating antibodies and pMHC tetramers. This suggests an absence of 'inactive-active' conformational states in the TCR constant regions and the extracellular CD3 subunits, unlike the transmembrane regions of the complex. This study contributes a nuanced understanding of TCR signaling, which may inform the development of therapeutics for immune-related disorders.
Competing Interests: Disclosure and competing interests statement. AN declares no competing interests. MK serves on the scientific advisory boards of Genentech and Merck and Co. and received research support from Merck Sharp & Dohme Corp., a subsidiary of Merck and Co., Inc., Genentech, Biogen, Novartis and the Mark Foundation.
(© 2024. The Author(s).)