Distinct conformational states of SARS-CoV-2 spike protein

Abstract

Intervention strategies are urgently needed to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. The trimeric viral spike (S) protein catalyzes fusion between viral and target cell membranes to initiate infection. Here, we report two cryo-electron microscopy structures derived from a preparation of the full-length S protein, representing its prefusion (2.9-angstrom resolution) and postfusion (3.0-angstrom resolution) conformations, respectively. The spontaneous transition to the postfusion state is independent of target cells. The prefusion trimer has three receptor-binding domains clamped down by a segment adjacent to the fusion peptide. The postfusion structure is strategically decorated by N-linked glycans, suggesting possible protective roles against host immune responses and harsh external conditions. These findings advance our understanding of SARS-CoV-2 entry and may guide the development of vaccines and therapeutics.

Fig. 1. Preparation of a full-length SARS-CoV-2 spike protein.

(A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S) protein. Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail; and tree-like symbols for glycans. A strep-tag was fused to the C terminus of S protein by a flexible linker. (B) The purified S protein was resolved by gel-filtration chromatography on a Superose 6 column in the presence of detergent NP-40. The molecular weight standards include thyoglobulin (670 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). Three major peaks (peak I-III) contain the S protein. (C) Load sample and peak fractions from (B) were analyzed by Coomassie stained SDS-PAGE. Labeled bands were confirmed by Western blot (S, S1 and S2) or protein sequencing (S2 and Cont; S and S1 bands did not gave any meaningful results probably due to a blocked N terminus). Cont, copurified contaminating protein, identified as endoplasmic reticulum chaperone BiP precursor by N-terminal sequencing. *, a putative S1/S2-S2’ fragment. Representative images and 2D averages by negative stain EM of three peak fractions are also shown. The box size of 2D averages is ~510Å.

Fig. 2. Cryo-EM structure of the SARS-CoV-2 S protein in the prefusion conformation.

(A) The structure of the S trimer was modeled based on a 2.9Å density map. Three protomers (A, B, and C) are colored in green, blue and red, respectively. (B) Overall structure of S protein in the prefusion conformation shown in ribbon representation. Various structural components in the color scheme shown in Fig. 1A include NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; and CD, connector domain. N terminus, S1/S2 cleavage site and S2’ cleavage site are indicated.

Fig. 3. Selected new features of the SARS-CoV-2 prefusion S trimer.

(A) N-terminal segment of S protein. The N terminus is at residue Gln14 after cleavage of the signal peptide. Cys15 forms a disulfide bond with Cys136. We observed good density for the N-linked glycan at Asn17. (B) A segment immediately downstream of the fusion peptide, while disordered in the stabilized soluble S ectodomain trimer structure, forms a tightly packed structure, designated FPPR for the fusion peptide proximal region, abutting CTD1. The newly identified FPPR structure would clash with CTD1 in the RBD up conformation. Various domains are shown in the color scheme in Fig. 2B. The structure of the soluble S trimer with one RBD in the up conformation (PDB ID: 6vyb) is shown in gray. In the box, a close-up view of the FPPR with adjacent fusion peptide in both surface representation and stick model. (C) The SARS-CoV-2 prefusion S trimer, viewed along the threefold axis, is superposed on the structure of the stabilized soluble S ectodomain trimer in the closed conformation with all three RBDs in the down conformation (PDB ID: 6vxx). While the S2 region is well aligned, there is a significant shift (e.g., ~12Å between two Ala123 residues) in S1. (D) Impact of the proline mutations introduced at residues 986 and 987 to stabilize the prefusion conformation. K986P mutation removes a salt bridge between Lys986 of one protomer and either Asp427 or Asp428 of another protomer in the trimer interface.

Fig. 4. Cryo-EM structure of the SARS-CoV-2 S2 in the postfusion conformation.

(A) The structure of the S2 trimer was modeled based on a 3.3Å density map. Three protomers (A, B, and C) are colored in green, blue and red, respectively. (B) Overall structure of the S2 trimer in the postfusion conformation shown in ribbon diagram. Various structural components in the color scheme shown in Fig. 1A include HR1, heptad repeat 1; CH, central helix region; CD, connector domain; and HR1, heptad repeat 2. The S2’ cleavage site is in a disordered loop between Ile770 and Thr912. Possible locations of the S2 N terminus (S1/S2 cleavage site), the FP and FPPR are also indicated. (C) A low-resolution map showing the density pattern for 5 N-linked glycans, with almost equal spacing along the long axis.

Fig. 5. A model for structural rearrangements of SARS-Cov-2 S protein.

(A) Structural changes independent of a target cell. We suggest that both the prefusion and postfusion spikes are present on the surface of mature virion and the ratio between them may vary (diagram of virion). The postfusion spikes on the virion are formed by S2 after S1 dissociates in the absence of ACE2. (B) ACE2-dependent structural rearrangements. Structural transition from the prefusion to postfusion conformation inducing membrane fusion likely proceeds stepwise as follows: 1) FPPR clamps down RBD through CTD1 in the prefusion S trimer (this study), but it occasionally flips out of position and allows an RBD to sample the up conformation (PDB ID: 6vyb). 2) RBD binding to ACE2 (PBD ID: 6m17) creates a flexible FPPR that enables exposure of the S2’ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2’ site, and perhaps also the S1/S2 site, releases the structural constraints on the fusion peptide and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. 3) Formation of the long central three-stranded coiled-coil and folding back of HR2. 4) Formation of the postfusion structure of S2 (this study) that brings the two membranes together, facilitating formation of a fusion pore and viral entry.