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Structural principles of B cell antigen receptor assembly

Nature volume 612, pages 156–161 (2022)Cite this article

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Abstract

The B cell antigen receptor (BCR) is composed of a membrane-bound class M, D, G, E or A immunoglobulin for antigen recognition1,2,3 and a disulfide-linked Igα (also known as CD79A) and Igβ (also known as CD79B) heterodimer (Igα/β) that functions as the signalling entity through intracellular immunoreceptor tyrosine-based activation motifs (ITAMs)4,5. The organizing principle of the BCR remains unknown. Here we report cryo-electron microscopy structures of mouse full-length IgM BCR and its Fab-deleted form. At the ectodomain (ECD), the Igα/β heterodimer mainly uses Igα to associate with Cµ3 and Cµ4 domains of one heavy chain (µHC) while leaving the other heavy chain (µHC′) unbound. The transmembrane domain (TMD) helices of µHC and µHC′ interact with those of the Igα/β heterodimer to form a tight four-helix bundle. The asymmetry at the TMD prevents the recruitment of two Igα/β heterodimers. Notably, the connecting peptide between the ECD and TMD of µHC intervenes in between those of Igα and Igβ to guide TMD assembly through charge complementarity. Weaker but distinct density for the Igβ ITAM nestles next to the TMD, suggesting potential autoinhibition of ITAM phosphorylation. Interfacial analyses suggest that all BCR classes utilize a general organizational architecture. Our studies provide a structural platform for understanding B cell signalling and designing rational therapies against BCR-mediated diseases.

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Fig. 1: Cryo-EM maps of the IgM BCR.
Fig. 2: ECD Interactions between the Igα/β heterodimer and mIgM.
Fig. 3: CP interactions between the Igα/β heterodimer and mIgM.
Fig. 4: TMD assembly of the IgM BCR.

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Data availability

All data and materials reported in the main and supplementary data are available upon reasonable request. The electron density maps of 8.2 Å full-length IgM BCR, 3.3 Å IgM BCRΔfab and 3.9 Å IgM BCRΔfab have been deposited in the Electron Microscopy Data Bank (EMDB) with accession codes EMD-27848, EMD-27888 and EMD-28030, respectively. The atomic coordinates for IgM BCRΔfab and full-length IgM BCR have been deposited in the Protein Data Bank with the accession code of 8E4C and 8EMA.

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Acknowledgements

We thank members of the Wu laboratory, especially Y. Zheng, for helpful discussions; R. Walsh, S. Sterling. M. Mayer and S. Rawson at the Harvard Cryo-EM Center for Structural Biology for cryo-EM training and data collection; K. Song at the University of Massachusetts Cryo-EM Core for screening and preliminary dataset collection; and J. Myers, V. Rayaprolu and the Pacific Northwest Center for Cryo-EM at Oregon Health and Science University for preliminary dataset collection, under the NIH grant U24GM129547 and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. We thank SBGrid for software and computing support. Research reported in this publication was supported by the National Institutes of Health through RO1 grant AI145656 (to M.R.), the DFG through TRR130-P02 (to M.R.), Germany’s Excellence Strategy (CIBSS-EXC-2189, Project ID390939984) and the Charles A. King Trust postdoctoral research fellowship (to Y.D.).

Author information

Author notes

  1. These authors contributed equally: Ying Dong and Xiong Pi

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Ying Dong, Xiong Pi & Hao Wu

  2. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA, USA

    Ying Dong, Xiong Pi, Zhuoyi Liang, Frederick W. Alt & Hao Wu

  3. Signaling Research Centers BIOSS and CIBSS, Freiburg, Germany

    Frauke Bartels-Burgahn, Deniz Saltukoglu, Jianying Yang & Michael Reth

  4. Department of Molecular Immunology, Faculty of Biology, University of Freiburg, Freiburg, Germany

    Frauke Bartels-Burgahn, Deniz Saltukoglu, Jianying Yang & Michael Reth

  5. HHMI, Boston Children’s Hospital, Boston, MA, USA

    Zhuoyi Liang & Frederick W. Alt

  6. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Zhuoyi Liang & Frederick W. Alt

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Contributions

M.R. and H.W. conceptualized the project. F.B.-B., D.S. and J.Y. made the stable cell lines for BCR expression. Z.L. validated BCR expression by flow cytometry under the supervision of F.W.A. Y.D. resorted and grew the cells, and purified the BCR samples for negative staining and cryo-EM. Y.D. and X.P. performed cryo-EM data acquisition. X.P. and Y.D. performed data processing. Y.D. and X.P. built and refined the model. Y.D. made the figures. H.W., Y.D. and M.R. wrote the manuscript with input from all authors.

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Correspondence to Michael Reth or Hao Wu.

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Extended data figures and tables

Extended Data Fig. 1 Protein purification and cryo-EM data processing of full-length IgM BCR.

a, Selection of J558L B cells that expressed IgM BCR. Domain organization of IgM BCR and surface expression of IgM (APC) and Igα (GFP) by flow cytometry are shown. b-c, Purification of IgM BCR shown by SDS-PAGE, Blue-native (BN) PAGE and western blotting using antibodies against the individual subunits. H: heavy chain; L: light chain. d, Gel filtration profiles of IgM BCR. The peak of IgM BCR is shaded in grey. e-f, Representative 2D classes of IgM BCR (e) and specifically at its Fab region (f). g-h, Data processing flow chart (g) and local resolution distribution of IgM BCR (h).

Extended Data Fig. 2 Protein purification, raw images and 2D classifications of IgM BCRΔFab.

a, Selection of J558L B cells that expressed IgM BCRΔFab. Domain organization of IgM BCRΔFab and surface expression of IgM (APC) and Igα (GFP) by flow cytometry are shown. b-c, Purification of IgM BCRΔFab shown by SDS-PAGE, Blue-native (BN) PAGE and western blotting using antibodies against the individual subunits. H: heavy chain. d, Gel filtration profiles of IgM BCRΔFab. The peak of IgM BCRΔFab is shaded in grey e-f, Representative negative staining EM image (e) and cryo-EM image of IgM BCRΔFab (f). g, Representative 2D classes of IgM BCRΔFab.

Extended Data Fig. 3 Cryo-EM data processing of IgM BCRΔFab.

a, Cryo-EM data processing flow chart of the 3.9 Å intermediate cryo-EM map and the 3.3 Å final cryo-EM map of IgM BCRΔFab. b, Angular distribution of the particles used for the final reconstruction shown as a heat map. c, Fourier shell correlation (FSC) plots. d, 3D FSC plot.

Extended Data Fig. 4 AlphaFold predicted models of IgM BCR.

a, The top ranked model of IgM BCR (without Fab and Cµ2) predicted by AlphaFold, coloured by per-residue pLDDT score. b, Five predicted models of IgM BCR. c, Alignment of the five models of the Igα/β heterodimer at ECD (left) and TMD (right), showing consistent prediction of the ECD interaction (left) and the TMD interaction (right) separately. The CPs of the Igα/β heterodimer were not predicted correctly. d, Alignment of the five models of mIgM at ECD (left) and TMD (right) superimposed with IgM BCRΔFab model, showing consistent prediction of the ECD interaction (left) but incorrect prediction of the TMD interaction (right). The CPs of the mIgM dimer were not predicted correctly. In (c) and (d), the experimentally determined subunits of the IgM BCRΔFab model are shown in their model colours and AlphaFold predicted models are shown in grey.

Extended Data Fig. 5 Model fitting of IgM BCRΔFab in the 3.3 Å cryo-EM map (contour level: 4.0 σ).

a, Ig regions of Igα and Igβ for the Igα/β interaction superimposed with the cryo-EM map. b, Interface between µHC Cµ4 and Igα superimposed with the cryo-EM map. c, Four connecting peptide regions from µHC, µHC’, Igα and Igβ superimposed with the cryo-EM map. Acidic and basic residues are shown and labelled. d-e, TMD helices of µHC, µHC’, Igα and Igβ superimposed with the cryo-EM map (d). Key residues mediating the TMD interaction are shown (e). f, The fitting of five glycosylation sites on the Igα/Igβ heterodimer.

Extended Data Fig. 6 Structural and sequence alignment of Igα and Igβ.

a, Structural alignment between the Ig domains of Igα and Igβ (3.2 Å RMSD). b-c, Sequence alignment of Igα (b) and Igβ (c) among different species. Residues at the interface of Igα/β with µHC and µHC’ are indicated by triangle symbols. Igα C113 and Igβ C135 are marked by red triangle symbols.

Extended Data Fig. 7 Structural and sequence alignment of mIg.

a, Structural alignment of the Cµ3-Cµ4 homodimer of IgM BCR with that from the crystal structure of secreted IgM or sIgM (3.1 Å RMSD, PDB: 6KXS). b, Sequence alignment of mIg among different isotypes. Residues at the interface of µHC and µHC’ with Igα/β are indicated by solid and hollow triangle symbols respectively.

Extended Data Fig. 8 Interaction at TMD and sequence alignment of mIgM.

a—e, The interactions between pairs of helices of TMD. f, Sequence alignment of mIgM among different species.

Extended Data Fig. 9 AlphaFold prediction and secondary structure prediction of Igβ, and sequence conservation mapping on the IgM-BCR surface.

a, AlphaFold prediction of Igβ. The TMD and ICD of Igβ were predicted, showing the pLDDT scores. b, Secondary structure prediction of Igβ (residue 171–228). The ITAM consensus motif is highlighted by red rectangle. c-d, Conserved residue distribution on the Cµ3-Cµ4 homodimer surface (c) and the Igα/Igβ heterodimer surface (d) according to sequence alignment among species. The most conserved residues are shown in dark blue, less conserved residues in light blue, and the remaining residues in their chain colours are defined in Fig. 1d.

Extended Data Table 1 Data collection, processing, refinement, and validation statistics
Full size table

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Dong, Y., Pi, X., Bartels-Burgahn, F. et al. Structural principles of B cell antigen receptor assembly. Nature 612, 156–161 (2022). https://doi.org/10.1038/s41586-022-05412-7

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