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. 2020 Dec;588(7837):327-330.
doi: 10.1038/s41586-020-2772-0. Epub 2020 Sep 17.

Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion

Donald J Benton #  1 , Antoni G Wrobel #  2 , Pengqi Xu  3   4 , Chloë Roustan  5 , Stephen R Martin  6 , Peter B Rosenthal  7 , John J Skehel  6 , Steven J Gamblin  8
Affiliations

Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion

Donald J Benton et al. Nature. 2020 Dec.

Abstract

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptors1-4, followed by fusion of the virus and cell membranes to release the virus genome into the cell. Both receptor binding and membrane fusion activities are mediated by the virus spike glycoprotein5-7. As with other class-I membrane-fusion proteins, the spike protein is post-translationally cleaved, in this case by furin, into the S1 and S2 components that remain associated after cleavage8-10. Fusion activation after receptor binding is proposed to involve the exposure of a second proteolytic site (S2'), cleavage of which is required for the release of the fusion peptide11,12. Here we analyse the binding of ACE2 to the furin-cleaved form of the SARS-CoV-2 spike protein using cryo-electron microscopy. We classify ten different molecular species, including the unbound, closed spike trimer, the fully open ACE2-bound trimer and dissociated monomeric S1 bound to ACE2. The ten structures describe ACE2-binding events that destabilize the spike trimer, progressively opening up, and out, the individual S1 components. The opening process reduces S1 contacts and unshields the trimeric S2 core, priming the protein for fusion activation and dissociation of ACE2-bound S1 monomers. The structures also reveal refolding of an S1 subdomain after ACE2 binding that disrupts interactions with S2, which involves Asp61413-15 and leads to the destabilization of the structure of S2 proximal to the secondary (S2') cleavage site.

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Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

Extended Data Fig. 1
Extended Data Fig. 1. Surface representation of obtained structures.
The three monomers of S in each trimer are coloured in blue, rosy brown and gold with ACE2 shown in green. Relative percentages of all trimeric S particles used to calculate electron microscopy maps are shown.
Extended Data Fig. 2
Extended Data Fig. 2. Features of the obtained spike structures.
a, Two three-dimensional classes, obtained by further classification of the one-ACE2-bound closed state from Fig. 1, representative of the range of motion of the RBD with bound ACE2, tilting away from the trimer axis of the spike trimer. The tilt of the RBD and ACE2 is indicated with a dashed line. b, Representative density of different obtained electron microscopy maps for residues 996–1030 of S2. Built model shown in pink, with EM density shown as a mesh. c, d, Comparison of spike structures for the open one-erect-RBD structure (purple) with the one-ACE2-bound structure (orange). c, S1 domains shown to highlight domain shifts of the RBD and RBD-associated intermediate domain. d, Outwards movements of spike domains (excluding RBDs). e, Comparison of RBD displacements of one-bound, two-bound and three-bound RBDs after binding of ACE2 to the unbound open structure of the spike protein (beige). These are compared to the RBD displacement after binding of the C105 Fab fragment, which binds at the ACE2 interface of the RBD (PDB: 6XCM).
Extended Data Fig. 3
Extended Data Fig. 3. Cryo-electron microscopy data processing scheme.
Classes of particles used to obtain the final spike trimer structures, unbound and in complex with ACE2, are surrounded by a box of the same colour as the final maps shown at the bottom. The global resolution, final particle number and percentage for each trimer species are shown at the bottom.
Extended Data Fig. 4
Extended Data Fig. 4. Monomeric S1 bound to ACE2.
a, Classification scheme for the S1–ACE2 complex. b, c, Maps are shown of orthogonal views of the non-uniform refinement (b) and unmasked refinement (c) of the final particles. Domains are coloured as follows: green, ACE2; yellow, NTD; rosy brown, RBD; pink, RBD ganymede; blue, NTD ganymede; cream, disseminated S1 density in b.
Extended Data Fig. 5
Extended Data Fig. 5. Fourier shell correlation graphs for each of the determined structures.
FSC, Fourier shell correlation.
Extended Data Fig. 6
Extended Data Fig. 6. Maps and models of determined structures.
Top, orthogonal views of electron microscopy density (grey) and ribbon diagram representation of the models. Bottom, electron microscopy maps coloured by local resolution shown below.
Fig. 1
Fig. 1. Sequential steps in ACE2 binding of the SARS-CoV-2 spike protein.
Surface representation of the spike, with monomers coloured in blue, rosy brown and gold, and ACE2 coloured in green. Each step shows two views of the spike complexes: a trimer axis vertical view (left) and an orthogonal top-down view along the axis (right). Clockwise from the top, we show structures for closed, open but unbound RBD, followed by sequential ACE2-binding events until reaching the fully open, three-ACE2-bound spike protein state. From this final trimeric species, we show dissociation into monomeric S1–ACE2, which may also occur for the one- or two-ACE2-bound species.
Fig. 2
Fig. 2. Structural rearrangements between the closed and the ACE2-bound states of the spike protein.
a, Surface representation of a monomer of S2 in the one-ACE2-bound, two-RBD-closed state coloured in light pink with the S1 subunit of the adjacent monomer in ribbon representation; the S1 of the one-ACE2-bound, two-RBD-closed state is shown in green and the three-RBD-closed state (PDB 6ZGE) is shown in blue. The atoms on the surface of S2 that contact the S1 intermediate domains are coloured in red. The arrows indicate the direction of movements of the intermediate domains, and of the RBD, between the closed and ACE2-bound conformations of the spike. b, Ribbon representations of the NTD-associated intermediate domain in blue and the moiety of the S2 chain that it interacts with (in red) in the closed conformation of the spike. Essential residues that participate in the interaction are labelled; of particular note is the salt bridge between Asp614 (S1, chain A) and Lys854 (S2, chain B). c, Ribbon representation of the same intermediate domain as in b, but in the conformation observed in the ACE2-bound structure of the spike (in green), in which the movement and refolding of the domain leads to a loss of interaction with S2, which becomes disordered. The putative fusion peptide (FP) and the S2′ site of the second protease cleavage at R815 adjacent to the region that undergoes unfolding are shown in dark red.
Fig. 3
Fig. 3. Structural basis of S2 unsheathing by ACE2 binding.
The spike protein is shown as a space-filling representation for S1, with each monomer coloured blue, rosy brown and gold, and as a ribbon representation for S2 coloured in red for all three monomers. Left, top-down and side-on views of the trimer in the closed conformation. Right, the same views for the fully open three-ACE2-bound species.
Fig. 4
Fig. 4. ACE2-bound S1 subunit as a part of the spike trimer and as an isolated monomer.
Space-filling representations of the spike protein with one monomer coloured polychromatically. NTD, yellow; NTD-associated subdomain, blue; RBD-associated subdomain, pink; RBD, rosy brown; S2, red; ACE2, green. The remainder of the trimer on the left is coloured grey. The structure on the right is aligned on the RBD:ACE2 moiety of the trimer complex on the left. The arrow indicates the direction of movement of the NTD and NTD-associated subdomain on the transition from the trimer (left) to the monomer species (right).
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