Structure-guided covalent stabilization of coronavirus spike glycoprotein trimers in the closed conformation

Abstract

SARS-CoV-2 is the causative agent of the COVID-19 pandemic, with 10 million infections and more than 500,000 fatalities by June 2020. To initiate infection, the SARS-CoV-2 spike (S) glycoprotein promotes attachment to the host cell surface and fusion of the viral and host membranes. Prefusion SARS-CoV-2 S is the main target of neutralizing antibodies and the focus of vaccine design. However, its limited stability and conformational dynamics are limiting factors for developing countermeasures against this virus. We report here the design of a construct corresponding to the prefusion SARS-CoV-2 S ectodomain trimer, covalently stabilized by a disulfide bond in the closed conformation. Structural and antigenicity analyses show we successfully shut S in the closed state without otherwise altering its architecture. We demonstrate that this strategy is applicable to other β-coronaviruses, such as SARS-CoV and MERS-CoV, and might become an important tool for structural biology, serology, vaccine design and immunology studies.

Fig. 1. Structure-based engineering of a SARS-CoV-2 S trimer in the closed conformation.

a,b, Cryo-EM structures of SARS-CoV-2 S with one S^B receptor-binding domain open (a, PDB 6VYB) and one in the closed state (b, PDB 6VXX), used as a basis for the design of intermolecular disulfide bonds. c, Pairs of residues mutated to create potential disulfide bonds are shown with dashed black lines between the Cα In panels a–c, each S protomer is colored distinctly. d, SDS–PAGE analysis in reducing and nonreducing conditions showing formation of an intermolecular disulfide bond. βME, β-mercaptoethanol. The uncropped image is shown in Supplementary Data 1. e,f, Electron micrograph of negatively stained SARS-CoV-2 2P DS S confirming proper folding of the designed protein construct (e) and representative two-dimensional class averages (f).

Fig. 2. Cryo-EM structure of the closed SARS-CoV-2 2P DS S glycoprotein.

a, Cryo-EM map of the SARS-CoV-2 2P DS S trimer in the closed conformation at 2.9-Å resolution. b, Ribbon diagram of the SARS-CoV-2 2P DS S trimer atomic model in the same orientation as in panel a. In panels a and b, each S protomer is colored distinctly. Asterisks show the locations of the introduced disulfide bonds. c, Superimposition of the SARS-CoV-2 2P DS S trimer (green) to the coordinates from the 2.8-Å SARS-CoV-2 2P S structure in the closed conformation, PDB 6VXX (ref. ) (black). d, Enlarged view of the designed disulfide bond with the corresponding region of cryo-EM density shown as a blue mesh.

Fig. 3. Evaluation of SARS-CoV-2 2P DS S antigenicity.

a–d, Binding to immobilized SARS-CoV-2 2P DS S (green) or SARS-CoV-2 2P S (black) of serially diluted concentrations of the human neutralizing antibodies S309 (a), S2H14 (c) and S304 (d) and the human ACE2 receptor fused to human Fc (b). e, Neutralization of SARS-CoV-2 S pseudovirus with human serum obtained from a patient with COVID-19. f, Binding of a serial dilution of the neutralizing convalescent serum shown in panel e to immobilized SARS-CoV-2 2P DS S (green) or SARS-CoV-2 2P S (black). Data are shown as mean and s.d. of n = 2 technical replicates; data are representative of two independent experiments. Data behind all graphs are available in Supplementary Data 1. A₄₅₀, absorbance at 450 nm; ND, not determined.

Fig. 4. Evaluation of SARS-CoV-2 2P DS S thermal stability and protease resistance.

a,b, Electron microscopy analysis of negatively stained SARS-CoV-2 2P S (a) and SARS-CoV-2 2P DS S (b) incubated for 20 min at 25, 55 and 85 °C. Black arrows highlight particles that appear to be misfolded. Red arrows highlight particles that appear to be in the postfusion conformation. c–e, Binding of human neutralizing antibody S309 to immobilized SARS-CoV-2 2P DS S (green) or SARS-CoV-2 2P S (black) preincubated for 20 min at 25, 55 and 85 °C (c), or for 16 h at 4 °C with 1, 10 or 100 µg ml⁻¹ trypsin (d) or chymotrypsin (e). Graphs show the area under the curve of binding of serially diluted concentrations of the human neutralizing antibody S309; data are shown as mean and s.d. of n = 2 technical replicates, and are representative of one (d and e) or two (c) independent experiments. Data behind graphs are available in Supplementary Data 1.

Fig. 5. Design and validation of SARS-CoV 2P DS S and MERS-CoV 2P DS S.

a, Sequence alignment showing the conservation of the residues involved in and adjacent to the designed disulfide bond across human coronavirus S glycoproteins. Residues are highlighted if they are identical in the alignment (black) or conservatively substituted (gray). Residues are numbered according to the SARS-CoV-2 S sequence. Green triangles highlight residues involved in the designed disulfide bond. b, SDS–PAGE analysis of MERS-CoV 2P S and MERS-CoV 2P DS S in reducing and nonreducing conditions showing formation of an intermolecular disulfide bond. c, 3D reconstruction in two orthogonal orientations of negatively stained MERS-CoV 2P DS S confirming proper folding of the designed protein construct. d, SDS–PAGE analysis of SARS-CoV 2P S and SARS-CoV 2P DS S in reducing and nonreducing conditions showing formation of an intermolecular disulfide bond. e, 3D reconstruction in two orthogonal orientations of negatively stained SARS-CoV 2P DS S confirming proper folding of the designed protein construct. f,g, Binding of various concentrations of the human neutralizing antibodies S309 (f) and S304 (g) to immobilized SARS-CoV 2P DS S (green) or SARS-CoV 2P S (black). Data are shown as mean and s.d. of n = 2 technical replicates; data are representative of two independent experiments.

Extended Data Fig. 1. CryoEM data processing and validation.

a. Local resolution map calculated using cryoSPARC. b-c. Representative electron micrograph (c) and class averages (b) of SARS-CoV-2 2P DS S embedded in vitreous ice. Scale bar: 100 nm. d. Gold-standard Fourier shell correlation curves. The 0.143 cutoff is indicated by an horizontal blue line. e. Particle orientation distribution plot.

Extended Data Fig. 2. Orthogonal views of the classes obtained by 3D classification.

Percentages reflect the proportion of particles that classified with each map.

Extended Data Fig. 3. Cryo-EM structure of the closed SARS-CoV-2 DS S glycoprotein.

Zoomed-in view of the designed disulfide bond with the corresponding region of cryo-EM density shown as a blue mesh.