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Structural basis for Fc receptor recognition of immunoglobulin M

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Abstract

Immunoglobulin Fc receptors are cell surface transmembrane proteins that bind to the Fc constant region of antibodies and play critical roles in regulating immune responses by activation of immune cells, clearance of immune complexes and regulation of antibody production. FcμR is the immunoglobulin M (IgM) antibody isotype-specific Fc receptor involved in the survival and activation of B cells. Here we reveal eight binding sites for the human FcμR immunoglobulin domain on the IgM pentamer by cryogenic electron microscopy. One of the sites overlaps with the binding site for the polymeric immunoglobulin receptor (pIgR), but a different mode of FcμR binding explains its antibody isotype specificity. Variation in FcμR binding sites and their occupancy reflects the asymmetry of the IgM pentameric core and the versatility of FcμR binding. The complex explains engagement with polymeric serum IgM and the monomeric IgM B-cell receptor (BCR).

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Fig. 1: Multivalent binding of FcμR to pentameric IgM.
Fig. 2: Different occupancies of FcμR among eight binding sites.
Fig. 3: Structure of FcμR Ig-like domain and in comparison with pIgR-D1.
Fig. 4: The binding interface between FcμR and IgM.
Fig. 5: Distributions of interacting residues on the Fc receptors and the targeted Ig molecules.

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Data availability

The structural data that support the findings of this study have been deposited in the Protein Data Bank and EM Data bank. The 8:1 FcμR/IgM-Fc model displayed in Fig. 1 has entry number EMD-16150 and PDB 8BPE. The IgM-Fc core with one FcμR at subunit Fcμ1 has entry number EMD-16151 and PDB 8BPF. The IgM subunit Fcμ3 with two FcμR has entry number EMD-16152 and PDB 8BPG. Source data are provided with this paper.

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Acknowledgements

We thank A. Nans of the Structural Biology Science Technology Platform for advice on data collection and computing; A. Purkiss and P. Walker of the Structural Biology Science Technology Platform; the Scientific Computing Science Technology Platform for computational support. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (CC2106 (P.B.R.), CC2006 (P.T.)), the UK Medical Research Council (CC2106 (P.B.R.), CC2006 (P.T.)) and the Wellcome Trust (CC2106 (P.B.R.), CC2006 (P.T.)). For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.

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Authors and Affiliations

  1. Structural Biology Science Technology Platform, The Francis Crick Institute, London, UK

    Qu Chen & Laura Masino

  2. Immune Receptor Activation Laboratory, The Francis Crick Institute, London, UK

    Rajesh P. Menon & Pavel Tolar

  3. Institute of Immunity and Transplantation, University College London, London, UK

    Pavel Tolar

  4. Structural Biology of Cells and Viruses Laboratory, The Francis Crick Institute, London, UK

    Peter B. Rosenthal

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  1. Qu Chen
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Contributions

Q.C., R.P.M. and L.M. performed experiments. Q.C., R.P.M., L.M., P.T. and P.B.R. contributed to experimental design, data analysis and manuscript writing.

Corresponding authors

Correspondence to Pavel Tolar or Peter B. Rosenthal.

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Nature Structural & Molecular Biology thanks Brian Sutton and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Editor recognition statement: Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Binding of Full-length IgM and IgM-Fc core to surface-immobilised FcμR.

(a) Structure schematic for full-length IgM. (b) Structure schematic for proteolysed IgM-Fc core. (c) Binding of full-length (green) and proteolysed IgM-Fc core (blue) to FcμR monitored by Bio-Layer Interferometry (BLI). Representative data sets for each form of IgM are shown. Instrument response values are plotted against IgM concentration. Fitting curves are shown as black lines. The apparent equilibrium dissociation constants (Kd) for full-length IgM and IgM-Fc core are 0.3 ± 0.1 nM and 0.7 ± 0.1 nM respectively. Technical replicates gave values of 0.4 ± 0.1 nM and 0.7 ± 0.1 nM respectively. Raw data for the plot and the technical replicates are provided in the Source Data file.

Source data

Extended Data Fig. 2 Single particle analysis of FcμR/IgM-Fc.

(a) Flow chart of the data processing for both non-tilted and tilted datasets. Steps are conducted in Relion 3.1 (clear box) or Cryosparc 3.2.0 (grey box). (b) A typical micrograph with three particles highlighted in red dotted circles. (c) Typical 2D classes of the complex. (d) 3D auto-refined map. (e) Half-map Fourier shell correlation (FSC) plot showing 3.5 Å global resolution.

Extended Data Fig. 3 Cryo-EM structure of IgM-Fc/FcμR complex.

(a) Particle subset selection by focused 3D classification at the IgM-Fc core and map refinement. (b) Front and side view of the complex model. IgM in grey, FcμR in rainbow colours. (c) Fitting of model and map shown in (a) and (b). (d) Fourier shell correlation (FSC) with 3.6 Å resolution at 0.143 cut-off and map-model FSC plot showing 3.9 Å resolution at 0.5 cut-off calculated with the model shown in (b) calculated in Phenix. (e) Local resolution of the refined map calculated in Cryosparc. (f) Eulerian angle distribution of the particles in the non-uniform refinement. (g) 3DFSC histogram calculated in Cryosparc of the refined map showing anisotropy between 3.2 Å −5.4 Å.

Extended Data Fig. 4 Maps of focused 3D classification at all IgM subunits Fcμ1 to Fcμ5 for quantification of FcμR occupancy at each subunit.

The central EM density is the refined map shown in Extended Data Fig. 3, reconstructed with 516,875 particles. Mask used for focused 3D classification for each subunit (containing Cμ4 dimer, Cμ3 dimer and FcμR) is shown in a specific colour (subunit Fcμ1, red; subunit Fcμ2, orange; subunit Fcμ3, yellow; subunit Fcμ4, green; subunit Fcμ5, pink). The 3D classes show different FcμR binding states (at front, back, both or neither) at each subunit individually. The front and the back of IgM is shown at the top-left of the figure using the same definition described in the main text.

Extended Data Fig. 5 Cryo-EM structure of FcμR/IgM-Fc complex focused on subunit Fcμ1.

(a) Particle subset selection by focused 3D classification at subunit Fcμ1 and map refinement. (b) Front and side view of the complex model. IgM in grey, FcμR in red. (c) Fitting of model and map shown in (a) and (b). (d) Fourier shell correlation (FSC) with 3.5 Å resolution at 0.143 cut-off and map-model FSC plot showing 3.6 Å resolution at 0.5 cut-off calculated in Phenix. (e) Local resolution of the refined map calculated in Cryosparc. (f) Eulerian angle distribution of the particles in the non-uniform refinement. (g) 3DFSC histogram of the refined map calculated in Cryosparc of the refined map showing anisotropy between 3.2 Å −7.6 Å.

Extended Data Fig. 6 Cryo-EM structure of FcμR/IgM-Fc complex focused on subunit Fcμ3.

(a) Particle subset selection by focused 3D classification at subunit Fcμ3 and map refinement. (b) Front and side view of the complex model. IgM in grey, FcμR in yellow. (c) Fitting of model and map shown in (a) and (b). (d) Fourier shell correlation (FSC) with 3.1 Å resolution at 0.143 cut-off and map-model FSC plot showing 3.3 Å resolution at 0.5 cut-off calculated in Phenix. (e) Local resolution of the refined map calculated in Cryosparc. (f) Eulerian angle distribution of the particles in the non-uniform refinement. (g) 3DFSC histogram calculated in Cryosparc of the refined map showing angular resolution distribution from 3.0 Å − 3.8 Å.

Extended Data Fig. 7 Density maps of key regions at FcμR and FcμR/IgM binding interface.

(a–b) The two conserved disulfide bonds in FcμR. (c–g) Densities of the interacting residues on FcμR and Cμ4 domains, corresponding to the interactions shown in Fig. 4b. FcμR in dark yellow, Cμ4-B chain in light grey, and Cμ4-A chain in slate grey. (h) Densities of the residues in CDR3 region of FcμR interacting with the neighbouring Cμ4 domain (in brown), corresponding to Fig. 4c. (i) Densities of the residues in CDR3 regions of FcμR interacting with the tailpiece of Fcμ5 chain (in pink), corresponding to Fig. 4d.

Extended Data Fig. 8 N-linked glycosylation at Asn563 contacting FcμR at subunit Fcμ3.

(a) The tailpiece assembly of the IgM pentamer showing ten N-linked glycosylation sites (purple). (b) The map of subunit Fcμ3 (same map as Extended Data Fig. 6a, before postprocessing, map threshold = 0.2). (c) Zoom-in view of the N-Acetylglucosamine (NAG) molecules (orange) linking from Asn563 (purple) at the tailpiece of Fcμ3B chain to FcμR-3f (yellow). (d) The overall map of FcμR/IgM-Fc (same map as Extended Data Fig. 3a, before postprocessing, map threshold = 0.2). (e) Cross-section of the map in (d) indicated by the red dotted line showing the densities of the two NAG chains (orange arrowheads) extending from Asn563 of the two Fcμ chains (Fcμ3A and Fcμ3B) to the two FcμR molecules at both sides.

Extended Data Table 1 Buried surface areas (BSA) between the individual CDR loops of the receptors and the immunoglobulin binding partner
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Supplementary information

Source data

Source Data Extended Data Fig. 1

Raw data for the BLI experiments.

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Chen, Q., Menon, R.P., Masino, L. et al. Structural basis for Fc receptor recognition of immunoglobulin M. Nat Struct Mol Biol 30, 1033–1039 (2023). https://doi.org/10.1038/s41594-023-00985-x

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