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

• Published in: EMBO reports
• Main Authors: Natarajan, Aswin, Velmurugu, Yogambigai, Becerra Flores, Manuel, Dibba, Fatoumatta, Beesam, Saikiran, Kikvadze, Sally, Wang, Xiaotian, Wang, Wenjuan, Li, Tianqi, Shin, Hye Won, Cardozo, Timothy, Krogsgaard, Michelle
• Format: Journal Article
• Language: English
• Published: 07-11-2024
• ISSN: 1469-3178; 1469-3178
• DOI: 10.1038/s44319-024-00314-3

Abstract 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.