In Situ Cell-surface Conformation of the TCR-CD3 Signaling Complex

Overview

Journal EMBO Rep

Specialty Molecular Biology

Date 2024 Nov 7

PMID 39511422

Authors

Aswin Natarajan

Yogambigai Velmurugu

Manuel Becerra Flores

Fatoumatta Dibba

Saikiran Beesam

Sally Kikvadze

Xiaotian Wang

Wenjuan Wang

Tianqi Li

Hye Won Shin

Timothy Cardozo

Michelle Krogsgaard

Affiliations

Soon will be listed here.

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.

References

Arechaga I, Swamy M, Abia D, Schamel W, Alarcon B, Valpuesta J . Structural characterization of the TCR complex by electron microscopy. Int Immunol. 2010; 22(11):897-903. DOI: 10.1093/intimm/dxq443. View

Arnett K, Harrison S, Wiley D . Crystal structure of a human CD3-epsilon/delta dimer in complex with a UCHT1 single-chain antibody fragment. Proc Natl Acad Sci U S A. 2004; 101(46):16268-73. PMC: 528977. DOI: 10.1073/pnas.0407359101. View

Beckett D, Kovaleva E, Schatz P . A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 1999; 8(4):921-9. PMC: 2144313. DOI: 10.1110/ps.8.4.921. View

Beddoe T, Chen Z, Clements C, Ely L, Bushell S, Vivian J . Antigen ligation triggers a conformational change within the constant domain of the alphabeta T cell receptor. Immunity. 2009; 30(6):777-88. DOI: 10.1016/j.immuni.2009.03.018. View

Birnbaum M, Berry R, Hsiao Y, Chen Z, Shingu-Vazquez M, Yu X . Molecular architecture of the αβ T cell receptor-CD3 complex. Proc Natl Acad Sci U S A. 2014; 111(49):17576-81. PMC: 4267357. DOI: 10.1073/pnas.1420936111. View

Zhong S, Malecek K, Perez-Garcia A, Krogsgaard M . Retroviral transduction of T-cell receptors in mouse T-cells. J Vis Exp. 2010; (44). PMC: 3185635. DOI: 10.3791/2307. View

Borroto A, Mallabiabarrena A, Albar J, Martinez-A C, Alarcon B . Characterization of the region involved in CD3 pairwise interactions within the T cell receptor complex. J Biol Chem. 1998; 273(21):12807-16. DOI: 10.1074/jbc.273.21.12807. View

Call M, Pyrdol J, Wiedmann M, Wucherpfennig K . The organizing principle in the formation of the T cell receptor-CD3 complex. Cell. 2003; 111(7):967-79. PMC: 3420808. DOI: 10.1016/s0092-8674(02)01194-7. View

Call M, Wucherpfennig K . The T cell receptor: critical role of the membrane environment in receptor assembly and function. Annu Rev Immunol. 2005; 23:101-25. DOI: 10.1146/annurev.immunol.23.021704.115625. View

Chang-Gonzalez A, Mallis R, Lang M, Reinherz E, Hwang W . Asymmetric framework motion of TCRαβ controls load-dependent peptide discrimination. Elife. 2024; 13. PMC: 10869138. DOI: 10.7554/eLife.91881. View

10.

Chen Y, Zhu Y, Li X, Gao W, Zhen Z, Dong D . Cholesterol inhibits TCR signaling by directly restricting TCR-CD3 core tunnel motility. Mol Cell. 2022; 82(7):1278-1287.e5. DOI: 10.1016/j.molcel.2022.02.017. View

11.

Coin I . Application of non-canonical crosslinking amino acids to study protein-protein interactions in live cells. Curr Opin Chem Biol. 2018; 46:156-163. DOI: 10.1016/j.cbpa.2018.07.019. View

12.

Coin I, Katritch V, Sun T, Xiang Z, Siu F, Beyermann M . Genetically encoded chemical probes in cells reveal the binding path of urocortin-I to CRF class B GPCR. Cell. 2013; 155(6):1258-69. PMC: 3916339. DOI: 10.1016/j.cell.2013.11.008. View

13.

Das D, Feng Y, Mallis R, Li X, Keskin D, Hussey R . Force-dependent transition in the T-cell receptor β-subunit allosterically regulates peptide discrimination and pMHC bond lifetime. Proc Natl Acad Sci U S A. 2015; 112(5):1517-22. PMC: 4321250. DOI: 10.1073/pnas.1424829112. View

14.

Davis M, Bjorkman P . T-cell antigen receptor genes and T-cell recognition. Nature. 1988; 334(6181):395-402. DOI: 10.1038/334395a0. View

15.

Dong D, Zheng L, Lin J, Zhang B, Zhu Y, Li N . Structural basis of assembly of the human T cell receptor-CD3 complex. Nature. 2019; 573(7775):546-552. DOI: 10.1038/s41586-019-1537-0. View

16.

Fernandes R, Shore D, Vuong M, Yu C, Zhu X, Pereira-Lopes S . T cell receptors are structures capable of initiating signaling in the absence of large conformational rearrangements. J Biol Chem. 2012; 287(16):13324-35. PMC: 3339974. DOI: 10.1074/jbc.M111.332783. View

17.

Fernandez-Recio J, Totrov M, Abagyan R . Soft protein-protein docking in internal coordinates. Protein Sci. 2002; 11(2):280-91. PMC: 2373434. DOI: 10.1110/ps.19202. View

18.

Fernandez-Recio J, Totrov M, Abagyan R . ICM-DISCO docking by global energy optimization with fully flexible side-chains. Proteins. 2003; 52(1):113-7. DOI: 10.1002/prot.10383. View

19.

Gagnon L, Cao Y, Cho A, Sedki D, Huber T, Sakmar T . Genetic code expansion and photocross-linking identify different β-arrestin binding modes to the angiotensin II type 1 receptor. J Biol Chem. 2019; 294(46):17409-17420. PMC: 6873184. DOI: 10.1074/jbc.RA119.010324. View