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

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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.

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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.


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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

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.

Correspondence to Ning Gao or Zhiwei Huang.

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The authors declare no competing interests.

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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.

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