Abstract
Immunoglobulin M (IgM) is the first antibody to emerge during embryonic development and the humoral immune response1. IgM can exist in several distinct forms, including monomeric, membrane-bound IgM within the B cell receptor (BCR) complex, pentameric and hexameric IgM in serum and secretory IgM on the mucosal surface. FcμR, the only IgM-specific receptor in mammals, recognizes different forms of IgM to regulate diverse immune responses2,3,4,5. However, the underlying molecular mechanisms remain unknown. Here we delineate the structural basis of the FcμR–IgM interaction by crystallography and cryo-electron microscopy. We show that two FcμR molecules interact with a Fcμ-Cμ4 dimer, suggesting that FcμR can bind to membrane-bound IgM with a 2:1 stoichiometry. Further analyses reveal that FcμR-binding sites are accessible in the context of IgM BCR. By contrast, pentameric IgM can recruit four FcμR molecules to bind on the same side and thereby facilitate the formation of an FcμR oligomer. One of these FcμR molecules occupies the binding site of the secretory component. Nevertheless, four FcμR molecules bind to the other side of secretory component-containing secretory IgM, consistent with the function of FcμR in the retrotransport of secretory IgM. These results reveal intricate mechanisms of IgM perception by FcμR.
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Data availability
Cryo-EM density maps of FcμR–Fcμ–J and FcμR–Fcμ–J–SC have been deposited in the Electron Microscopy Data Bank with accession codes EMD-34085 (1:1), EMD-34086 (4:1) and EMD-34074. Structural coordinates have been deposited in the PDB with the accession codes 7YTC, 7YTD and 7YSG. The crystal structure of FcμR-D1–Fcμ-Cμ4 has been deposited in the PDB with the accession code 7YTE. The cryo-EM structure of Fcμ–J–SC was determined previously and is available from the PDB under the accession code 6KXS. Source data are provided with this paper.
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Acknowledgements
We thank the staff of the National Facility for Protein Science Shanghai (beamline BL19U) for assistance with X-ray data collection; the Core Facilities at the School of Life Sciences, Peking University for help with negative-staining EM; the Cryo-EM Platform of Peking University for help with data collection; the High-performance Computing Platform of Peking University for help with computation; and the National Center for Protein Sciences at Peking University for assistance with the Biacore, confocal microscopy and flow cytometry facilities. This work was partly supported by the Qidong-SLS Innovation Fund to J.X. and by Changping Laboratory. Y.L. was supported by the China Postdoctoral Innovative Talent Support Program (Boxin Plan, BX20200009).
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Contributions
Y.L., H.S. and R.Z. contributed equally to this work. Y.L. and H.S. carried out the crystallography and cryo-EM studies, as well as the SPR analyses. Y.L. performed confocal microscopy. R.Z. carried out flow cytometry and immunoprecipitation. Y.L., H.S. and R.Z. performed the pull-down experiments. C.J., Y.W. and C.S. provided critical assistance to these studies. J.X. conceived and supervised the project, and wrote the manuscript with input from all authors.
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Extended data figures and tables
Extended Data Fig. 1 SPR, coimmunoprecipitation, and pull-down experiments.
a. Two repeats of the SPR experiments. The SPR experiments were performed by passing over immobilized FcμR-D1 with two-fold serial dilutions of purified Cμ4, Fcμ–J, anti-CD20 IgM, or Fcμ–J–SC, and the highest concentration values are indicated in the graphs. The original SPR sensorgrams are shown in colored lines, whereas the fitted models are overlaid on the sensorgrams and shown in black. The sensorgrams in the first repeat are shown in the main figures. The model fitting statistics are also shown. b. Coimmunoprecipitation of FcμR with mIgM. Results of two independent experiments are shown. c. Repeat of the pull-down experiment presented in Fig. 4.
Extended Data Fig. 2 Sequence alignments of antibody Fc sequences and FcμR.
a. Sequence alignment of human antibody Fc sequences. Fcμ residues that are recognized by FcμR are highlighted with magenta rectangles. b. Sequence comparison between human FcμR and the mouse protein.
Extended Data Fig. 3 Purification of the FcμR-ECD–Fcμ–J and FcμR-ECD–Fcμ–J–SC complexes for cryo-EM.
a. Size-exclusion chromatography of the FcμR-ECD–Fcμ–J complex on a Superose 6 Increase column. The elution volumes of molecular weight markers are indicated. b. SDS–PAGE analyses of the FcμR-ECD–Fcμ–J complex. For gel source data, see Supplementary Fig. 1. All the purification experiments and corresponding SDS-PAGE analyses in this paper have been repeated at least two times with similar results. c. Size-exclusion chromatography of the FcμR-ECD–Fcμ–J–SC complex. d. SDS–PAGE analyses of the FcμR-ECD–Fcμ–J–SC complex. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 4 Cryo-EM 3D reconstruction of the FcμR-ECD–Fcμ–J complex.
a. A representative raw cryo-EM image out of 14,899 similar micrographs used for data processing. b. 2D classifications. c. Flow chart for image processing. d. Gold standard Fourier shell correlation (FSC) curves with estimated resolutions of the 1:1 FcμR-ECD–Fcμ–J complex. e. FSC curves of the 4:1 FcμR-ECD–Fcμ–J complex. f. Euler angle distribution of the classified particles for the 1:1 FcμR-ECD–Fcμ–J complex. g. Euler angle distribution of the classified particles for the 4:1 FcμR-ECD–Fcμ–J complex. h. Resolution estimations of the final map of the 1:1 FcμR-ECD–Fcμ–J complex. i. Resolution estimations of the final map of the 4:1 FcμR-ECD–Fcμ–J complex. j. FcμR-D1 interacts with the C-terminal region of the J-chain at the R1 site. k. FcμR-D1 interacts with the tailpiece of Fcμ5B at the R1 site. l. At the R2–R4 sites, in addition to mainly binding to the Cμ4 domains of Fcμ2B, Fcμ3B, and Fcμ4B, FcμR also interacts slightly with the C–C′ loop of Fcμ1B, Fcμ2B, and Fcμ3B from the adjoining Fcμ units. Shown here is a copy of the FcμR-D1–Fcμ-Cμ4 pair from the crystal structure (green) superposed to Fcμ2B, illustrating the close contact between the C–C′ loop of Fcμ1B and the R2 FcμR.
Extended Data Fig. 5 Cryo-EM analyses of the FcμR-D1–Fcμ–J complex.
a. A representative raw cryo-EM image of the FcμR-D1–Fcμ–J complex (prepared in a 10:1 molar ratio) out of 580 similar micrographs used for data processing. b. 2D classifications of the sample in a. c. Flow chart for image processing of the sample in a. d. FSC curves with estimated resolutions of the sample in a. e. A representative raw cryo-EM image of the FcμR-D1–Fcμ–J complex (prepared in a 200:1 molar ratio) out of 277 similar micrographs used for data processing. f. 2D classifications of the sample in e. g. Flow chart for image processing of the sample in e. h. FSC curves with estimated resolutions of the sample in e. i. 3D reconstruction of the FcμR-D1–Fcμ–J complex prepared in a 10:1 molar ratio. j. 3D reconstruction of the FcμR-D1–Fcμ–J complex prepared in a 200:1 molar ratio.
Extended Data Fig. 6 Cryo-EM 3D reconstructions of the FcμR-ECD–Fcμ–J–SC complex.
a. A representative raw cryo-EM image out of 6,066 similar micrographs used for data processing. b. 2D classifications. c. Flow chart for image processing. d. Gold standard Fourier shell correlation (FSC) curves with estimated resolutions. e. Euler angle distribution of the classified particles. f. Resolution estimations of the final map. g. Arg112FcμR contacts J-chain Ser65J–Asp66J as well as Glu570 in the tailpiece of Fcμ1A.
Extended Data Fig. 7 Gating strategy for the detection of FcμR expression and IgM binding by flow cytometry.
a. Gating strategy used to sort HEK293T cells for analyzing the cell surface FcμR levels. The strategy used for the WT FcμR expressing cells is shown, and the R45A/F67A mutant expressing cells were analyzed using the same strategy as WT. Cells stably expressing GFP were used as negative controls (Cont.) to set the level of background. b. Gating strategy used to sort HEK293T cells for analyzing IgM binding. c. Gating strategy used to sort Jurkat cells for analyzing the cell surface FcμR levels. d. Gating strategy used to sort Jurkat cells for analyzing IgM binding.
Extended Data Fig. 8 The binding of FcμR molecules at the R1–R4 sites may reduce the binding of FcμR on the other side.
a. FcμR-D1–Fcμ-Cμ4 crystal structures were superposed onto Fcμ2–5 in the 4:1 FcμR–Fcμ–J cryo-EM structure to generate a model with 4 molecules of FcμR bound also at the R1′–R4′ sites (gray). This 8:1 FcμR–Fcμ–J model was then aligned to the FcμR–Fcμ–J–SC cryo-EM structure based on the central tailpiece region and J-chain. The R1–R4 and R1′–R4′ FcμR molecules from the cryo-EM structures are shown in green and yellow, respectively. The Fcμ5 molecules are shown in two shades of blue, whereas the rest Fcμ molecules are shown in light yellow. J-chain and SC are shown in magenta and pink. The presence of FcμR at the R1–R4 side appears to render the Fcμ–J platform slightly concave; and the R1′–R4′ binding sites in the 4:1 FcμR–Fcμ–J structure are slightly displaced compared to the corresponding sites in the FcμR–Fcμ–J–SC complex. b. Two different views of the aligned structures in a to highlight the structural differences at the R1′–R4′ sites. When compared to the R1′–R4′ FcμR molecules (yellow) in the cryo-EM structure, all four docked FcμR molecules (gray) tilt towards the center of the Fcμ–J platform in the model. A simplified cartoon illustrating these differences is shown below (SC is not depicted in the cartoon). c. An enlarged view of the Fcμ5 molecules from the above aligned structures is shown to illustrate the moderate displacement of the R1′ site.
Supplementary information
Supplementary Figure 1
Raw data for gels and blots: a, original source image for Extended Data Fig. 2b. The molecular weight is marked; b, original source image for Extended Data Fig. 2d; c-f, original source images for Fig. 4a; g-j, original source images for Fig. 4b; k-o, original source images for Fig. 4c. Panels k and m represent two independent gels and blots from the same experiment, with the exposure time longer in m to make the FcμR-D1 bands clearer; p-r, original source images for Extended Data Fig. 1b (left panel); s, original source images for the left panel of extended Fig. 1c; t, original source images for the middle panel of extended Fig. 1c; u, original source images for the right panel of extended Fig. 1c.
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Li, Y., Shen, H., Zhang, R. et al. Immunoglobulin M perception by FcμR. Nature 615, 907–912 (2023). https://doi.org/10.1038/s41586-023-05835-w
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DOI: https://doi.org/10.1038/s41586-023-05835-w
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