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. 2018 Aug 13;14(8):e1007236.
doi: 10.1371/journal.ppat.1007236. eCollection 2018 Aug.

Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2

Wenfei Song  1 , Miao Gui  2 , Xinquan Wang  1   3 , Ye Xiang  2
Affiliations

Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2

Wenfei Song et al. PLoS Pathog. .

Abstract

The trimeric SARS coronavirus (SARS-CoV) surface spike (S) glycoprotein consisting of three S1-S2 heterodimers binds the cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediates fusion of the viral and cellular membranes through a pre- to postfusion conformation transition. Here, we report the structure of the SARS-CoV S glycoprotein in complex with its host cell receptor ACE2 revealed by cryo-electron microscopy (cryo-EM). The complex structure shows that only one receptor-binding domain of the trimeric S glycoprotein binds ACE2 and adopts a protruding "up" conformation. In addition, we studied the structures of the SARS-CoV S glycoprotein and its complexes with ACE2 in different in vitro conditions, which may mimic different conformational states of the S glycoprotein during virus entry. Disassociation of the S1-ACE2 complex from some of the prefusion spikes was observed and characterized. We also characterized the rosette-like structures of the clustered SARS-CoV S2 trimers in the postfusion state observed on electron micrographs. Structural comparisons suggested that the SARS-CoV S glycoprotein retains a prefusion architecture after trypsin cleavage into the S1 and S2 subunits and acidic pH treatment. However, binding to the receptor opens up the receptor-binding domain of S1, which could promote the release of the S1-ACE2 complex and S1 monomers from the prefusion spike and trigger the pre- to postfusion conformational transition.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Density maps of the ACE2-bound and ACE2-free SARS-CoV spikes.
Surface shadowed diagrams of the ACE2-bound conformation 1, the ACE2-bound conformation 2, the ACE2-bound conformation 3, unbound-up and unbound-down conformations of the SARS-CoV S glycoprotein after trypsin cleavage and low pH treatment. One CTD1 is colored pink and the bound ACE2 is colored green. The angle between the long axes of the CTD1 and the horizontal plane is shown at the bottom of each conformation.
Fig 2
Fig 2. Structural comparisons of the ACE2-bound and ACE2-free SARS-CoV spikes.
(A) Schematic and topology diagrams showing the domain organization. NTD, CTD3 and S2 are colored grey, CTD1 is colored red, CTD2 is colored orange, CTD1-CTD2 linkers are colored blue. (B) Cross-correlation coefficients (CCs) between the CTD2s or the S2s of different conformations. *Values in the parentheses are the CCs between the S2s of different conformations. Density maps were low-pass filtered to 5.5 Å and were compared at a contouring level of 4.0 σ. (C) Ribbon-diagram structural comparisons of the ACE2-bound conformation 3 and the unbound-down conformation. ACE2, CTD1, CTD2, CTD3 and S2 of the ACE2-bound conformation 3 are colored green, red, yellow, pink and pink respectively. The unbound-down conformation is colored grey. CTD2 and S2 domain are zoomed in to show the receptor-binding induced hinge motion of CTD2. (D) EM densities and corresponding atomic models represented in ribbon diagrams around the S2’ protease cleavage site: unbound-down conformation (left) and ACE2-bound conformation (right). The S2’ site is colored red and position of the S2’ site is indicated with black arrows. The fusion peptide is colored cyan. The “C” shape loop covering the S2’ site is colored blue.
Fig 3
Fig 3. Structure of the post-fusion SARS-CoV S2.
(A) A size-exclusion chromatography elution profile of the low pH and trypsin treated S and ACE2 mixture. (B) Negative staining analysis of the S2 peak. Red arrows indicate rosette-shape structures formed by the post-fusion S2 trimers. Scale bar: 50 nm. (C) 2D class averaged cryo-EM images of the SARS-CoV S2 rosette. Scale bar: 10 nm. (D) 3D density map of the SARS-CoV S2 in post-fusion state with a fitted SARS-CoV S2 homology model. The three protomers are colored pink, yellow and cyan, respectively. Left: bottom view; right: side view.
Fig 4
Fig 4. Structure of the disassociated S1-ACE2 complex.
(A) Size-exclusion chromatography profiles of ACE2 alone (blue), cleaved S (green) and the low pH and trypsin treated S and ACE2 mixture. (B) SDS-page analysis of the uncleaved S, cleaved S, S-ACE2, S1-ACE2 and ACE2 peak fractions in “A”. (C) 2D analysis of the S1-ACE2 peak in “A”. Upper: 2D projections of the ACE2 density map calculated from the atomic model (PDB ID: 2ajf). Middle: 2D class averaged images of the particles from the S1-ACE2 peak in “A”. Bottom: components of the complex marked in the 2D class averaged images. ACE2 and S1 densities are marked cyan and pink, respectively. (D) A 3D density map calculated from the particles of the S1-ACE2 peak. The CTD1, CTD2 (pink) and ACE2 (green) are fitted into the density map as a rigid body. The flexible NTD and CTD3 are not visible and are represented as an ellipse. (E) Ribbon diagrams showing the linker downstream the S1/S2 cleavage site and the S2’ cleavage site of ACE2-bound conformation 3 in S1 associated state (left) and S1 disassociated state (right), respectively. The S2’ cleavage site is colored red and indicated by a red arrow. The fusion peptide down-stream of the S2’ site is colored green and the “C” shape loop up-stream the S2’ site is colored blue. The S1 subunit and the linker down-stream the S1/S2 cleavage site of the adjacent protomer is colored cyan and pink, respectively. The black arrow indicates the flexibility of the linker after the disassociation of the S1 subunit.
Fig 5
Fig 5. The pre- to post-fusion structural transition of the S2 subunit.
Left: ribbon diagrams showing the prefusion SARS-CoV S2 structure in which the “down” CTD1 has direct contacts with the helix linker 2 (blue). H1: helix 1. H2: helix 2. H3: helix 3. H1, H2 and helix linker 1: cyan. Helix linker 2: blue. H3: dark green. CTD1: red. Connecting domain: pink. Middle: ribbon diagrams showing the S2 region of the S-ACE2 complex in which the “up” CTD1 has no direct contacts with the Helix linker 2. Right: ribbon diagram of the postfusion SARS-CoV S2. H1, H2, H3 and the linkers transit to form the long central helix. HR2: yellow.
Fig 6
Fig 6. A cartoon representation showing the pre- to post-fusion transition of the SARS-CoV S glycoprotein.
The “down” to “up” transition of the receptor-binding domain (CTD1) allows receptor binding. The binding to ACE2 opens up CTD1 and CTD2, promotes the disassociation of the S1-ACE2 complex from the S1/S2 cleaved S glycoprotein, induces the pre- to post-fusion transition of the S2 subunit, and initiates the membrane fusion.
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This work was supported by the National Key Plan for Scientific Research and Development of China (2016YFA0501100 and 2016YFD0500307), the National Natural Science Foundation of China (81471929, U1405228 and 31470721) and the Beijing Advanced Innovation Center for Structural Biology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.