Cryo-EM analysis of the post-fusion structure of the SARS-CoV spike glycoprotein

Abstract

Global emergencies caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 significantly endanger human health. The spike (S) glycoprotein is the key antigen and its conserved S2 subunit contributes to viral entry by mediating host-viral membrane fusion. However, structural information of the post-fusion S2 from these highly pathogenic human-infecting coronaviruses is still lacking. We used single-particle cryo-electron microscopy to show that the post-fusion SARS-CoV S2 forms a further rotated HR1-HR2 six-helix bundle and a tightly bound linker region upstream of the HR2 motif. The structures of pre- and post-fusion SARS-CoV S glycoprotein dramatically differ, resembling that of the Mouse hepatitis virus (MHV) and other class I viral fusion proteins. This structure suggests potential targets for the development of vaccines and therapies against a wide range of SARS-like coronaviruses.

Fig. 1. Overall structure of SARS-CoV S2 fusion machinery in post-fusion state.

a Schematic diagram of the SARS-CoV S2 subunit ectodomain. The uncolored region represents the undetermined region in our structure. L linker region, UH the upstream helices, FP fusion peptide, CR connecting region, HR heptad repeat, CH central helix, BH β-hairpin, SD3 subdomain 3. b–e Cartoon representation of the SARS-CoV S2 trimer in post-fusion state colored as shown in a.

Fig. 2. Structural and conformational changes of SARS-CoV S glycoprotein from pre-fusion to post-fusion state.

a Topology and cartoon representation of SARS-CoV S2 subunit in pre-fusion state (PDB: 6NB6) colored as in Fig. 1a. The S1 subunit in the pre-fusion state is colored white. b Topology and cartoon representation of SARS-CoV S2 subunit in post-fusion state colored as in Fig. 1a. Unobserved regions in the structures are colored in light gray or presented as dashed lines in the topology diagram of (a, b). c Structural transition of the HR1 and CH motifs during membrane fusion. d Structural changes of the UH, BH, and SD3 during membrane fusion.

Fig. 3. The linker region upstream of HR2 motif important for membrane fusion.

a Binding of the linker region upstream of HR2 motif along the central helix colored as in Fig. 1a. b The electrostatic surface representation of the linker region upstream of HR2 motif binding area. Red represents negative charge, and blue represents positive charge. c Overall structural changes of the linker region upstream of the HR2 motif during membrane fusion. The linker regions in pre-fusion (PDB: 6NB6) and post-fusion states are shown as cartoons colored in violet and green, respectively. The SD3 domains in pre-fusion (PDB: 6NB6) and post-fusion states are shown as cartoons colored in pale cyan and orange, respectively. d Structural changes of the linker region upstream of the HR2 motif during membrane fusion represented in single chain mode. The cartoons are colored as in (c). e Structural details of the linker region and the SD3 domain from the pre-fusion SARS-CoV (PDB: 6NB6) colored as in (c). f Structural details of the linker region and the SD3 domain from the post-fusion SARS-CoV colored as in (c). The disulfide bond is shown as spheres.

Fig. 4. Comparison of SARS-CoV and MHV S2 structures in post-fusion state.

a, b Cartoon representation of the comparison of post-fusion S2 overall structures from SARS-CoV (cyan) and MHV (PDB: 6B3O, wheat). c Superposition of SARS-CoV (colored as Fig. 1a) and MHV (PDB: 6B3O, wheat) post-fusion S2 subunits in single chain mode. d, e Structural comparison of the linker region upstream of HR2 motif from SARS-CoV (green) and MHV (PDB: 6B3O, wheat) shown as cartoon and electrostatic surface. Red represents negative charge, and blue represents positive charge.

Fig. 5. Glycan shield of SARS-CoV S2 fusion machinery in post-fusion state.

a Schematic diagram of the N-linked glycosylate modification sites of SARS-CoV S2 subunit. The visible N-linked glycosylation sites are shown in solid lines and the invisible N-linked glycosylation site is shown in dashed line. b–d The distribution of the glycan shield within the S2 subunit shown in side view (b), top view (c) and bottom view (d). The highly conserved glycosylation sites among coronavirus are shown as light blue spheres. The completely conserved glycosylation sites between SARS-CoV and SARS-CoV-2 are shown as yellow spheres.

Fig. 6. Potential therapeutic targets within SARS-CoV S2 subunit.

a Cartoon representation of the linker loop region upstream of the HR2 motif (blue loop), the HR1 motif (blue helix) and the HR2 motif (red). The residue D1128 is shown as spheres colored in red. b The U-turn loop connecting the HR1 and CH motif in the pre-fusion SARS-CoV S trimer (PDB: 5X 5B). The pre-fusion SARS-CoV structure is shown as cartoons colored in white. The U-turn loop is presented as spheres colored in red. c The U-turn loop and other loop regions within the HR1 and CH motifs in the pre-fusion S2 subunit (PDB: 5X 5B). The U-turn loop is shown as spheres colored in red and the other loop regions are shown as spheres colored in blue.