Structures of MERS-CoV spike glycoprotein in complex with sialoside attachment receptors

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Abstract

The Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and often lethal respiratory illness in humans, and no vaccines or specific treatments are available. Infections are initiated via binding of the MERS-CoV spike (S) glycoprotein to sialosides and dipeptidyl-peptidase 4 (the attachment and entry receptors, respectively). To understand MERS-CoV engagement of sialylated receptors, we determined the cryo-EM structures of S in complex with 5-N-acetyl neuraminic acid, 5-N-glycolyl neuraminic acid, sialyl-LewisX, α2,3-sialyl-N-acetyl-lactosamine and α2,6-sialyl-N-acetyl-lactosamine at 2.7–3.0 Å resolution. We show that recognition occurs via a conserved groove that is essential for MERS-CoV S-mediated attachment to sialosides and entry into human airway epithelial cells. Our data illuminate MERS-CoV S sialoside specificity and suggest that selectivity for α2,3-linked over α2,6-linked receptors results from enhanced interactions with the former class of oligosaccharides. This study provides a structural framework explaining MERS-CoV attachment to sialoside receptors and identifies a site of potential vulnerability to inhibitors of viral entry.

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Fig. 1: Cryo-EM identification of a sialoside-binding site in the MERS-CoV S glycoprotein.
Fig. 2: MERS-CoV S and HCoV-OC43 S glycoproteins interact with sialosides, using distinct binding grooves.
Fig. 3: The ligand-binding site is required for MERS-CoV S-mediated attachment to sialosides and entry into human airway epithelial cells.
Fig. 4: Structural basis of MERS-CoV S selectivity for α2,3-linked sialoside receptors.

Data availability

The cryo-EM maps (sharpened and unsharpened) and atomic models have been deposited to the EMDB and wwPDB under accession numbers EMD-20542 and PDB 6Q04 (MERS-CoV S + Neu5Ac), EMD-20829 (MERS-CoV S + Neu5Gc), EMD-20543 and PDB 6Q05 (MERS-CoV S + sLeX), EMD-20544 and PDB 6Q06 (MERS-CoV S + 2,3-SLN) and EMD-20545 and PDB 6Q07 (MERS-CoV S + 2,6-SLN). Source Data are available online for Extended Data Fig. 5.

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Acknowledgements

Research reported in this publication was supported by the National Institute of General Medical Sciences (R01GM120553, D.V.), the National Institute of Allergy and Infectious Diseases (HHSN272201700059C, D.V.), a Pew Biomedical Scholars Award (D.V.), an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund (D.V.) and a Swiss National Science Foundation PostDoc Mobility fellowship (M.M.S.). M.A.T. acknowledges support from the Institut Pasteur. This work was also supported by the Arnold and Mabel Beckman cryo-EM center at the University of Washington.

Author information

B.-J.B., F.D. and D.V. supervised the research. Y.-J.P., A.C.W., Z.W., M.M.S., W.L., M.A.T., B.-J.B. and D.V. designed the experiments. A.C.W. and M.M.S. expressed and purified the MERS-CoV S ectodomain. Y.-J.P. and Z.W. performed cryo-EM sample preparation and data collection. Y.-J.P., Z.W. and D.V. processed the cryo-EM data. Y.-J.P., Z.W., F.D. and D.V. built and refined the atomic models. A.C.W. and Z.W. performed S mutagenesis. A.C.W. carried out the pseudovirus assays. W.L. carried out the hemagglutination assays. Y.-J.P., A.C.W., W.L., B.-J.B. and D.V. analyzed the data. Y.-J.P., A.C.W. and D.V. prepared the manuscript with input from all authors.

Correspondence to David Veesler.

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Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Chemical structures of the sialosides used for structural studies.

a, Neu5Ac. b, Neu5Gc. c, sLeX. d, 2,3-SLN. e, 2,6-SLN.

Extended Data Fig. 2 CryoEM analysis of the MERS-CoV S glycoprotein in complex with Neu5Ac at 2.7 Å resolution.

a,b, Representative electron micrograph (a) and class averages (b) for the Neu5Ac-bound MERS-CoV S structure. c, Gold-standard (blue) and map/model (red) Fourier shell correlation curves. The 0.143 and 0.5 cutoffs are indicated by horizontal dashed lines. d, Local resolution map calculated using cryoSPARC. The Neu5Ac ligand is estimated to be resolved at 3Å resolution. eg, Representative cryoEM densities shown as blue mesh with the corresponding atomic model rendered as sticks colored grey, blue and red for carbon, nitrogen and oxygen atoms, respectively. Dashed bonds indicate hydrogen bonds with ordered water molecules.

Extended Data Fig. 3 Structural basis for the MERS-CoV S selectivity for neuraminic acids that are not 9-O-acetylated or 5-N-glycolylated.

a, Superimposition of the 5-N-acetyl,9-O-acetyl neuraminic acid α-methyl glycoside (9-O-Ac-Neu5Ac, green) from the HCoV-OC43 S holo structure (PDB 6NZK19) to the bound Neu5Ac from the MERS-CoV S/Neu5Ac complex structure suggests the MERS-CoV S sialoside-binding site could not sterically accommodate the 9-O-acetyl group in this conformation. The distances between the 9-O-acetyl group and His91 or Ala92 are indicated. b, The MERS-CoV S sialoside-binding site rendered as a ribbon diagram with the side chains of key surrounding residues shown. Neu5Ac is rendered with the corresponding region of cryoEM density from the MERS-CoV S/Neu5Ac complex structure (low-pass filtered to 3Å and scaled to the MERS-CoV S/Neu5Gc map) shown as a blue mesh contoured at 6σ. cf, The cryoEM structure of MERS-CoV S in complex with Neu5Gc shows weaker density for the sialoside (blue mesh contoured at 6σ), indicating poor steric and/or chemical accommodation of the 5-N-glycolyl hydroxyl group in the hydrophobic pocket defined by Phe39, Phe101, Ile131 and Ile132. In panels (c-e) Neu5Gc was modeled based on the porcine rotavirus CRW-8 VP8* holo structure (PDB 3TAY41) which corresponds to the conformation observed in 1 out of 8 structures available in the protein data bank with a Neu5Gc analogue. In panel f, Neu5Gc was modeled based on the rhesus rotavirus VP8* holo structure (PDB 3TB041) which is the only structure in the protein data bank featuring Neu5Gc with the 5-N-glycolyl group in an alternate orientation. The distance between the 5-N-glycolyl hydroxyl group and Gln36 is indicated.

Extended Data Fig. 4 CryoEM analysis of the MERS-CoV S glycoprotein in complex with Neu5Gc at 3.0 Å resolution.

a,b, Representative electron micrograph (a) and class averages (b) for the Neu5Gc-bound MERS-CoV S structure. c, Gold-standard Fourier shell correlation curve. The 0.143 cut-off is indicated by horizontal dashed lines. d, Representative density shown as blue mesh with the corresponding atomic model rendered as sticks colored grey, blue and red for carbon, nitrogen and oxygen atoms, respectively.

Extended Data Fig. 5 SDS-PAGE and Western blot analyses.

a, SDS-PAGE analysis of purified wild type or mutants MERS-CoV S domain A fused to human immunoglobulin Fc. Two micrograms of each protein was loaded. b, Western-blot analysis of murine leukemia viral particles pseudotyped with wild type or mutants MERS-CoV S using an anti-MERS-CoV S1 polyclonal antibody. Uncropped blot image is available as source data. Source data

Extended Data Fig. 6 CryoEM analysis of the MERS-CoV S glycoprotein in complex with sLeX at 2.7 Å resolution.

a,b, Representative electron micrograph (a) and class averages (b) for the sLeX-bound MERS-CoV S structure. c, Gold-standard (blue) and map/model (red) Fourier shell correlation curves. The 0.143 and 0.5 cutoffs are indicated by horizontal dashed lines. d, Representative density shown as blue mesh with the corresponding atomic model rendered as sticks colored grey, blue and red for carbon, nitrogen and oxygen atoms, respectively.

Extended Data Fig. 7 CryoEM analysis of the MERS-CoV S glycoprotein in complex with 2,3-SLN at 2.7 Å resolution.

a,b, Representative electron micrograph (a) and class averages (b) for the 2,3-SLN-bound MERS-CoV S structure. c, Gold-standard (blue) and map/model (red) Fourier shell correlation curves. The 0.143 and 0.5 cutoffs are indicated by horizontal dashed lines. d, Representative density shown as blue mesh with the corresponding atomic model rendered as sticks colored grey, blue and red for carbon, nitrogen and oxygen atoms, respectively.

Extended Data Fig. 8 CryoEM analysis of the MERS-CoV S glycoprotein in complex with 2,6-SLN at 2.9 Å resolution.

a,b, Representative electron micrograph (a) and class averages (b) for the 2,6-SLN-bound MERS-CoV S structure. c, Gold-standard (blue) and map/model (red) Fourier shell correlation curves. The 0.143 and 0.5 cutoffs are indicated by horizontal dashed lines. d, Representative density shown as blue mesh with the corresponding atomic model rendered as sticks colored grey, blue and red for carbon, nitrogen and oxygen atoms, respectively.

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Park, Y., Walls, A.C., Wang, Z. et al. Structures of MERS-CoV spike glycoprotein in complex with sialoside attachment receptors. Nat Struct Mol Biol 26, 1151–1157 (2019) doi:10.1038/s41594-019-0334-7

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