Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion

Abstract

The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. The coronavirus spike (S) glycoprotein initiates infection by promoting fusion of the viral and cellular membranes through conformational changes that remain largely uncharacterized. Here we report the cryoEM structure of a coronavirus S glycoprotein in the postfusion state, showing large-scale secondary, tertiary, and quaternary rearrangements compared with the prefusion trimer and rationalizing the free-energy landscape of this conformational machine. We also biochemically characterized the molecular events associated with refolding of the metastable prefusion S glycoprotein to the postfusion conformation using limited proteolysis, mass spectrometry, and single-particle EM. The observed similarity between postfusion coronavirus S and paramyxovirus F structures demonstrates that a conserved refolding trajectory mediates entry of these viruses and supports the evolutionary relatedness of their fusion subunits. Finally, our data provide a structural framework for understanding the mode of neutralization of antibodies targeting the fusion machinery and for engineering next-generation subunit vaccines or inhibitors against this medically important virus family.

Keywords: coronavirus; cryoEM; fusion proteins; membrane fusion; proteolytic activation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Proteolytic activation of coronavirus S proteins. (A) Schematic of the MHV S glycoprotein organization with emphasis on the S2 subunit. The dashed black box shows the region of the S2 polypeptide chain that is unresolved in the map. Gray dashed boxes show regions that were not part of the construct. BH, beta hairpin; CD, connector domain; CH, central helix; CT, cytoplasmic tail; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; SH, stem helix; TM, transmembrane domain; UH, upstream helix. (B–D) 2D class averages of negatively stained MHV S (B), SARS-CoV S (C), and MERS-CoV S (D) trimers in the prefusion state (Left) and in the trypsin-cleaved postfusion state (Right). (Scale bar, 10 nm.)

Fig. S1.

Characterization of the MHV S pre- to postfusion transition. (A–E) Annotated mass spectra showing that trypsin cleavage can occur at or near the predicted S1/S2 (A–C) and S2′ (D and E) sites. (F) Section of a micrograph with negatively stained MHV S2 rosettes formed upon incubation of prefusion S with 1:100 trypsin. (Scale bar, 10 nm.)

Fig. 2.

CryoEM structure of the MHV S2 postfusion machinery. (A) 3D map colored by protomer and viewed from the side with the fused membranes located at the top. (B) Ribbon diagram of the MHV S2 atomic model oriented as in A. (C and D) Ribbon diagram showing the atomic model from the extremity proximal (C) or distal (D) to the fused membranes.

Fig. S2.

CryoEM analysis of MHV S2. (A) Representative micrograph. (Scale bar, 10 nm.) (B) 2D class averages of frozen-hydrated MHV S2 particles. (Scale bar, 10 nm.) (C) Gold-standard (blue) and model/map (red) Fourier shell correlation curves. The 0.143 and 0.5 cutoff values are indicated by horizontal gray lines. The resolution was determined to be 4.1 Å.

Fig. S3.

Validation of the atomic model. (A and B) Two orthogonal views of the superimposition of the HR1/HR2 six-helix bundle structure derived from the MHV S2 cryoEM map (colored by protomer) and from X-ray crystallography (Protein Data Bank ID code 1WDF, gray). (C) Comparison of the core β-sheet structure in the prefusion (gray) and postfusion (colored as in Fig. 2) MHV S spike glycoprotein. (D) Comparison of the upstream helix structure in the prefusion (gray) and postfusion (colored as in Fig. 2) MHV S spike glycoprotein. (E and F) The MHV S2 model is shown in gray ribbon with the cryoEM density shown in transparent light gray. Disulfide bonds are shown in green for residues 775–797 (E) and 1,082–1,093 (F). (G and H) Glycans linked to Asn-1180 (G) and Asn-1190 (H) are rendered as ball and sticks with the corresponding region of the cryoEM map shown as a blue mesh. Carbon, nitrogen, and oxygen atoms are colored gray, blue, and red, respectively. (I and J) Mass spectra for Asn1180 (I) and Asn1190 (J) showing the presence of glycans at these glycosylation sequons.

Fig. 3.

Conformational changes associated with the fusion reaction. (A) Ribbon and topology diagrams of the MHV S2 subunit in the prefusion conformation (6). (B) Ribbon and topology diagrams of the MHV S2 subunit in the postfusion conformation. (C and D) Ribbon rendering of the MHV S central helix and HR1 in the prefusion (C) and postfusion (D) states highlighting the jack-knife refolding of the four HR1 helices and intervening regions into a single continuous helix.

Fig. 4.

Comparison of the coronavirus and paramyxovirus fusion machineries. (A and B) Postfusion structures of MHV S (A) and RSV F (B) with one protomer of each trimer colored yellow and the other two colored gray.

Fig. S4.

Comparison of the MHV S postfusion S2 structure with other fusion proteins. (A) Superimposition of the HCoV-NL63 S2 (gray) and MHV postfusion S2 (colored as in Fig. 2) subunits. Only the upstream and central helices, core β-sheet, and connector domain are shown. (B) Superimposition of the HCoV-NL63 S2 (gray) and MHV postfusion S2 (pink) connector domains emphasizing their structural conservation. (C and D) Topology diagrams comparing postfusion MHV S2 and RSV F glycoproteins. Conserved elements in the RSV F subunit are shown using the same colors; nonconserved elements are shown in gray.

Fig. S5.

Location of fusion-inhibiting mutations in MHV S. (A and B) Prefusion MHV S2 subunit shown in gray ribbon from the side (A) and from the viral membrane (B). (C and D) Postfusion MHV S2 shown in gray ribbon from the side (C) and looking toward the long axis (D). Two residues known to attenuate fusogenicity upon substitution are shown as red spheres.

Fig. 5.

Proposed model of coronavirus entry. (A) The S glycoprotein promotes virus attachment to a host cell via binding to a transmembrane receptor using either domain A (e.g., MHV S) or domain B (e.g., SARS-CoV or MERS-CoV S). The prefusion MHV S trimer is shown with the S1 subunit depicted in gray and the S2 subunits colored by protomer. (B) Upon receptor binding, activation of the S trimer occurs via protease cleavage at the S2′ site. (C) Shedding of the S1 subunit trimer frees the fusion machinery, as reported for MERS-CoV (10). (D) Subsequent conformational changes of the S glycoprotein result in fusion of the viral and host membranes. The postfusion MHV S2 trimer is depicted with each protomer in a different color. The transmembrane helices and the fusion peptides (FP) are connected to the MHV S trimer with dotted and solid lines, respectively.