Structural basis of assembly of the human T cell receptor–CD3 complex

Abstract

The αβ T cell receptor (TCR), in association with the CD3γε–CD3δε–CD3ζζ signalling hexamer, is the primary determinant of T cell development and activation, and of immune responses to foreign antigens. The mechanism of assembly of the TCR–CD3 complex remains unknown. Here we report a cryo-electron microscopy structure of human TCRαβ in complex with the CD3 hexamer at 3.7 Å resolution. The structure contains the complete extracellular domains and all the transmembrane helices of TCR–CD3. The octameric TCR–CD3 complex is assembled with 1:1:1:1 stoichiometry of TCRαβ:CD3γε:CD3δε:CD3ζζ. Assembly of the extracellular domains of TCR–CD3 is mediated by the constant domains and connecting peptides of TCRαβ that pack against CD3γε–CD3δε, forming a trimer-like structure proximal to the plasma membrane. The transmembrane segment of the CD3 complex adopts a barrel-like structure formed by interaction of the two transmembrane helices of CD3ζζ with those of CD3γε and CD3δε. Insertion of the transmembrane helices of TCRαβ into the barrel-like structure via both hydrophobic and ionic interactions results in transmembrane assembly of the TCR–CD3 complex. Together, our data reveal the structural basis for TCR–CD3 complex assembly, providing clues to TCR triggering and a foundation for rational design of immunotherapies that target the complex.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: 3D reconstruction and atomic model of the human TCR–CD3 complex.
Fig. 2: Structures of TCRαβ and CD3.
Fig. 3: Extracellular interfaces of the TCR–CD3 complex.
Fig. 4: Transmembrane assembly of the TCR–CD3 complex.
Fig. 5: Structural superimposition of TCR–CD3 with ligand- or antibody-engaged subunits.

Data availability

The atomic coordinates of TCR–CD3 have been deposited in the Protein Data Bank with the accession code 6JXR. The corresponding maps have been deposited in the Electron Microscopy Data Bank with the accession code EMD-9895. The datasets generated and analysed during the current study are available from the corresponding authors upon reasonable request.

References

  1. 1.

    Germain, R. N. & Stefanová, I. The dynamics of T cell receptor signaling: complex orchestration and the key roles of tempo and cooperation. Annu. Rev. Immunol. 17, 467–522 (1999).

    CAS  PubMed  Google Scholar 

  2. 2.

    Davis, M. M. & Bjorkman, P. J. T-cell antigen receptor genes and T-cell recognition. Nature 334, 395–402 (1988).

    ADS  CAS  PubMed  Google Scholar 

  3. 3.

    Samelson, L. E. & Klausner, R. D. The T-cell antigen receptor. Structure and mechanism of activation. Ann. NY Acad. Sci. 540, 1–3 (1988).

    ADS  CAS  PubMed  Google Scholar 

  4. 4.

    Rossjohn, J. et al. T cell antigen receptor recognition of antigen-presenting molecules. Annu. Rev. Immunol. 33, 169–200 (2015).

    CAS  PubMed  Google Scholar 

  5. 5.

    Meuer, S. C. et al. Evidence for the T3-associated 90K heterodimer as the T-cell antigen receptor. Nature 303, 808–810 (1983).

    ADS  CAS  PubMed  Google Scholar 

  6. 6.

    Wucherpfennig, K. W., Gagnon, E., Call, M. J., Huseby, E. S. & Call, M. E. Structural biology of the T-cell receptor: insights into receptor assembly, ligand recognition, and initiation of signaling. Cold Spring Harb. Perspect. Biol. 2, a005140 (2010).

    PubMed  PubMed Central  Google Scholar 

  7. 7.

    Gaud, G., Lesourne, R. & Love, P. E. Regulatory mechanisms in T cell receptor signalling. Nat. Rev. Immunol. 18, 485–497 (2018).

    CAS  PubMed  Google Scholar 

  8. 8.

    Rudolph, M. G., Stanfield, R. L. & Wilson, I. A. How TCRs bind MHCs, peptides, and coreceptors. Annu. Rev. Immunol. 24, 419–466 (2006).

    CAS  PubMed  Google Scholar 

  9. 9.

    Wang, J. H. & Reinherz, E. L. The structural basis of αβ T-lineage immune recognition: TCR docking topologies, mechanotransduction, and co-receptor function. Immunol. Rev. 250, 102–119 (2012).

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Call, M. E., Pyrdol, J. & Wucherpfennig, K. W. Stoichiometry of the T-cell receptor–CD3 complex and key intermediates assembled in the endoplasmic reticulum. EMBO J. 23, 2348–2357 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Garboczi, D. N. et al. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature 384, 134–141 (1996).

    ADS  CAS  PubMed  Google Scholar 

  12. 12.

    Bäckström, B. T. et al. A motif within the T cell receptor α chain constant region connecting peptide domain controls antigen responsiveness. Immunity 5, 437–447 (1996).

    PubMed  Google Scholar 

  13. 13.

    Wang, Y. et al. A conserved CXXC motif in CD3epsilon is critical for T cell development and TCR signaling. PLoS Biol. 7, e1000253 (2009).

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    Love, P. E. & Hayes, S. M. ITAM-mediated signaling by the T-cell antigen receptor. Cold Spring Harb. Perspect. Biol. 2, a002485 (2010).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Kane, L. P., Lin, J. & Weiss, A. Signal transduction by the TCR for antigen. Curr. Opin. Immunol. 12, 242–249 (2000).

    CAS  PubMed  Google Scholar 

  16. 16.

    van der Merwe, P. A. & Dushek, O. Mechanisms for T cell receptor triggering. Nat. Rev. Immunol. 11, 47–55 (2011).

    PubMed  Google Scholar 

  17. 17.

    Arnett, K. L., Harrison, S. C. & Wiley, D. C. Crystal structure of a human CD3-ε/δ dimer in complex with a UCHT1 single-chain antibody fragment. Proc. Natl Acad. Sci. USA 101, 16268–16273 (2004).

    ADS  CAS  PubMed  Google Scholar 

  18. 18.

    Kjer-Nielsen, L. et al. Crystal structure of the human T cell receptor CD3εγ heterodimer complexed to the therapeutic mAb OKT3. Proc. Natl Acad. Sci. USA 101, 7675–7680 (2004).

    ADS  CAS  PubMed  Google Scholar 

  19. 19.

    Le Nours, J. et al. Atypical natural killer T-cell receptor recognition of CD1d-lipid antigens. Nat. Commun. 7, 10570–10584 (2016).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Call, M. E. et al. The structure of the ζζ transmembrane dimer reveals features essential for its assembly with the T cell receptor. Cell 127, 355–368 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Borg, N. A. et al. The CDR3 regions of an immunodominant T cell receptor dictate the ‘energetic landscape’ of peptide–MHC recognition. Nat. Immunol. 6, 171–180 (2005).

    CAS  PubMed  Google Scholar 

  22. 22.

    Hogquist, K. A.OKT3 and H57-597: from discovery, to commercialization, to the clinic. J. Immunol. 197, 3429–3430 (2016).

    CAS  PubMed  Google Scholar 

  23. 23.

    Sadelain, M., Rivière, I. & Riddell, S. Therapeutic T cell engineering. Nature 545, 423–431 (2017).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    June, C. H., O’Connor, R. S., Kawalekar, O. U., Ghassemi, S. & Milone, M. C. CAR T cell immunotherapy for human cancer. Science 359, 1361–1365 (2018).

    ADS  CAS  PubMed  Google Scholar 

  25. 25.

    Yan, C. et al. Structure of a yeast spliceosome at 3.6-angstrom resolution. Science 349, 1182–1191 (2015).

    ADS  CAS  PubMed  Google Scholar 

  26. 26.

    Kastner, B. et al. GraFix: sample preparation for single-particle electron cryomicroscopy. Nat. Methods 5, 53–55 (2008).

    CAS  Google Scholar 

  27. 27.

    Beddoe, T. et al. Antigen ligation triggers a conformational change within the constant domain of the αβ T cell receptor. Immunity 30, 777–788 (2009).

    CAS  PubMed  Google Scholar 

  28. 28.

    Birnbaum, M. E. et al. Molecular architecture of the αβ T cell receptor–CD3 complex. Proc. Natl Acad. Sci. USA 111, 17576–17581 (2014).

    ADS  CAS  PubMed  Google Scholar 

  29. 29.

    Natarajan, A. et al. Structural model of the extracellular assembly of the TCR–CD3 complex. Cell Rep. 14, 2833–2845 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Call, M. E., Pyrdol, J., Wiedmann, M. & Wucherpfennig, K. W. The organizing principle in the formation of the T cell receptor–CD3 complex. Cell 111, 967–979 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Fernandes, R. A. et al. T cell receptors are structures capable of initiating signaling in the absence of large conformational rearrangements. J. Biol. Chem. 287, 13324–13335 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Baker, B. M., Gagnon, S. J., Biddison, W. E. & Wiley, D. C. Conversion of a T cell antagonist into an agonist by repairing a defect in the TCR/peptide/MHC interface: implications for TCR signaling. Immunity 13, 475–484 (2000).

    CAS  PubMed  Google Scholar 

  33. 33.

    Yin, Y., Wang, X. X. & Mariuzza, R. A. Crystal structure of a complete ternary complex of T-cell receptor, peptide–MHC, and CD4. Proc. Natl Acad. Sci. USA 109, 5405–5410 (2012).

    ADS  CAS  PubMed  Google Scholar 

  34. 34.

    Sasada, T. et al. Involvement of the TCR Cβ FG loop in thymic selection and T cell function. J. Exp. Med. 195, 1419–1431 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Natarajan, K. et al. An allosteric site in the T-cell receptor Cβ domain plays a critical signalling role. Nat. Commun. 8, 15260–15274 (2017).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Chakraborty, A. K. & Weiss, A. Insights into the initiation of TCR signaling. Nat. Immunol. 15, 798–807 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Google Scholar 

  38. 38.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

    CAS  Google Scholar 

  42. 42.

    Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  Google Scholar 

  43. 43.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  Google Scholar 

  44. 44.

    Buchan, D. W., Minneci, F., Nugent, T. C., Bryson, K. & Jones, D. T. Scalable web services for the PSIPRED Protein Analysis Workbench. Nucleic Acids Res. 41, W349–W357 (2013).

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    CAS  Google Scholar 

  46. 46.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Google Scholar 

  47. 47.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

    CAS  Google Scholar 

  48. 48.

    Hovmöller, S., Zhou, T. & Ohlson, T. Conformations of amino acids in proteins. Acta Crystallogr. D 58, 768–776 (2002).

    PubMed  Google Scholar 

Download references

Acknowledgements

We thank the Core Facilities at School of Life Science and Technology of Harbin Institute of Technology and the Core Facilities of Peking University School of Life Sciences for assistance with negative-staining electron microscopy, and the cryo-EM platform of Peking University for help with cryo-EM data collection. The computation was supported by the High-performance Computing Platform of Peking University. We thank J. Chai for critical reading of the manuscript. This research was funded by the National Natural Science Foundation of China grant no. 31825008 and 31422014 to Z.H.; 31725007 and 31630087 to N.G.; 31800630 to Y.Z.; 31700655 to N.L. and the Ministry of Science and Technology of China (2016YFA0500700 to N.G.).

Author information

Affiliations

Authors

Contributions

D.D., J.L., B.Z., L.Z., S.X. and Y.W. prepared the protein samples for negative staining and cryo-EM. D.D., J.L. and L.Z. performed negative staining. L.Z. and N.L. performed cryo-EM data acquisition and data processing. Y.Z. and N.G. built and refined the model. N.G. oversaw the cryo-EM and contributed to the manuscript preparation. Z.H. directed the project, oversaw biochemistry studies, cryo-EM sample preparation and model building and wrote the manuscript with input and support from all co-authors.

Corresponding authors

Correspondence to Ning Gao or Zhiwei Huang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Ellis L. Reinherz and Nikolaos G. Sgourakis for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 TCR–CD3 protein complex purification.

a, Shown here are the results of western blot analyses using antibodies against the subunits of TCR–CD3. The peak fractions from gel filtration were immunoblotted with the indicated antibodies. b, The native or cross-linked TCR–CD3 protein bands were separated by reducing SDS–PAGE and visualized by staining with Coomassie blue. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 2 Binding affinities between conformation-specific antibodies and the native or glutaraldehyde-treated TCR–CD3 complex.

ae, Binding affinities of native or glutaraldehyde-treated TCR–CD3 protein complex with activation antibodies OKT3 and UCHT1 and three antibodies targeting CD3γ, CD3δ and CD3ζ, measured by bio-layer interferometry. Green or blue curves are the experimental trace obtained from bio-layer interferometry experiments, and red curves are the best global fits to the data used to calculate the equilibrium-dissociation constant (Kd) values. Data are representative of three independent experiments. f, Negative staining of TCR–CD3 particles before (top) and after (bottom) cross-linking.

Extended Data Fig. 3 Cryo-EM image processing procedure.

a, A representative raw cryo-EM image. b, Two-dimensional class averages of the TCR–CD3 particles. c, Image-processing workflow of the TCR–CD3 particles. d, Gold-standard FSC curve of the final density map. e, Angular distribution of the TCR–CD3 particles in the final round of 3D refinement. f, Final local resolution estimation of the cryo-EM map.

Extended Data Fig. 4 Cryo-EM density map of the TCR-CD3 complex.

ah, Local density of eight well-resolved TCR–CD3 subunits. The last four residues (D309–G312) in the cytoplasmic tail of TCRβ are not well defined by cryoEM density. In ah, density maps are displayed with a similar threshold. in, Local density of all N-linked glycosylation regions in the final map. o, p, Local density of two representative extracellular regions of TCR–CD3 complex in the final map. q, r, Local density of two representative transmembrane regions of TCR–CD3 complex in the final map.

Extended Data Fig. 5 Structural comparison of free and pMHC-bound TCRαβ with that from the TCR–CD3 complex.

a, Structural comparison of free (PDB: 4X6B, green) and pMHC-bound (PDB: 4WWK, cyan) TCRαβ with that from TCR–CD3 complex. b, Structural comparison of the pMHC-bound TCRα and TCRβ chains (PDB: 3T0E) with cryo-EM structure of TCR–CD3. Per-residue backbone atom root mean square deviation (RMSD) values for the TCRα and TCRβ chains are listed.

Extended Data Fig. 6 Structure of CD3ζζ′.

a, The interface of the CD3ζζ homodimer. b, Structural comparison of the CD3ζζ homodimer (in TCR–CD3) with free CD3ζζ (PDB: 2HAC) shown in red.

Extended Data Fig. 7 Density maps of key interaction sites in the transmembrane and ECD regions.

ac, Enlarged view of interaction sites in the transmembrane regions (related to Fig. 4b–d). di, Enlarged view of interaction in the ECD regions (related to Fig. 3b–d). Residues are colour-coded as in Fig. 1.

Extended Data Fig. 8 Density maps of the conserved regions in the Cβ domain and interactions between the CP segments of Cα and Cβ domains.

a, Density map of the region near helix 3 (residues E153–K159) of the Cβ domain. b, Density map of the region near helix 4 (residues S216–N225) of the Cβ domain. cf, Density map of the interaction sites between the CP segment of Cα and the H3 and H4 helices of the Cβ domain.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

Supplementary information

Supplementary Figure

This file contains the uncropped gel blots for Extended Data Fig. 1a, b.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Dong, D., Zheng, L., Lin, J. et al. Structural basis of assembly of the human T cell receptor–CD3 complex. Nature 573, 546–552 (2019). https://doi.org/10.1038/s41586-019-1537-0

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing