Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM

Abstract

The recent outbreaks of SARS-CoV-2 pose a global health emergency. The SARS-CoV-2 trimeric spike (S) glycoprotein interacts with the human ACE2 receptor to mediate viral entry into host cells. We report the cryo-EM structures of a tightly closed SARS-CoV-2 S trimer with packed fusion peptide and an ACE2-bound S trimer at 2.7- and 3.8-Å resolution, respectively. Accompanying ACE2 binding to the up receptor-binding domain (RBD), the associated ACE2-RBD exhibits continuous swing motions. Notably, the SARS-CoV-2 S trimer appears much more sensitive to the ACE2 receptor than the SARS-CoV S trimer regarding receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of SARS-CoV-2. We defined the RBD T470-T478 loop and Y505 as viral determinants for specific recognition of SARS-CoV-2 RBD by ACE2. Our findings depict the mechanism of ACE2-induced S trimer conformational transitions from the ground prefusion state toward the postfusion state, facilitating development of anti-SARS-CoV-2 vaccines and therapeutics.

Fig. 1. A tightly closed conformation of SARS-CoV-2 S trimer.

(A and B) Cryo-EM map and model of SARS-CoV-2 S trimer in a tightly closed state, with three protomers shown in different color. (C) Close-up view of the model map fitting in the NTD and RBD regions of the S1 subunit, illustrating that most of the NTD region was well resolved. (D) Overlaid RBD structures of our S-closed (blue) with a cryo-EM structure of SARS-CoV-2 S in closed state (6VXX, gray), illustrating that the RBM S469-C488 loop was captured in our structure (indicated by dotted ellipsoid). (E) Top view of the overlaid structures as in (D) (left) and zoom-in views of specific domains, showing that there is a marked counterclockwise rotation in S1 especially in NTD, resulting in a twisted, tightly closed conformation. (F) Protomer interaction interface analysis by PISA. (G) Location of the captured FP fragment (in deep pink) within the S trimer (left) and one protomer. S1 and S2 subunits are colored steel blue and gold, respectively. (H) Model map fitting for the FP fragment. (I) Close-up view of the interactions between D614 from SD2 and FP, with the hydrogen bonds labeled in dotted lines and the L828-F855 region in FP in deep pink.

Fig. 2. The architecture of the SARS-CoV-2 S-ACE2 complex.

(A and B) Cryo-EM map and model of SARS-CoV-2 S-ACE2 complex. We named the RBD up protomer as protomer 1 (light green), and the other two RBD down ones as protomer 2 (royal blue) and protomer 3 (gold). ACE2 was colored in violet red. (C) Side and top views of the overlaid S-open (color) and S-closed (dark gray) structures, showing that in the open process, there is a 71.0° upward/outward rotation of RBD associated with a downward shift of SD1 in protomer 1. (D) Rotations of NTD and CH from the S-closed (gray) to the S-open (in color) state, with the NTD also showing a downward/outward movement (right). (E) Side view of the overlaid S-ACE2 (violet red) and S-open (light green) protomer 1 structures, showing that the angle between the long axis of RBD and the horizontal plane of S trimer reduces from the S-open to the S-ACE2. (F) Top and side views of the overlaid S-ACE2 (violet red) and S-open (color) RBD structures, showing the coordinated movements of RBDs. (G) Protomer interaction interface analysis of S-ACE2 by PISA. (H) Aromatic interactions between the core region of the up RBD-1 (green) and the RBM T470-F490 loop of the neighboring RBD-2 (blue). (I) Overlaid structures of S-ACE2 (gray) and S-closed (color, with the FP fragment in deep pink), indicating a downward shift of SD1 and most of the FP is missing in S-ACE2. Close-up view (right) of the potential clashes between the downward-shifted SD1 β34 and α8 helix of FP. (J) Population shift between the ACE2-unpresented and ACE2-presented S trimer samples.

Fig. 3. The T470-T478 loop and residue Y505 within RBM play important roles in the engagement of SARS-CoV-2 spike with receptor ACE2.

(A) The overall view of ACE2 (violet red) bound protomer 1 (light green) from our S-ACE2 structure, and zoom-in view of the interaction interface between ACE2 and RBD, with the key contacting elements T470-F490 loop and Q498-Y505 within RBM highlighted in black ellipsoid and blue ellipsoid, respectively. (B) Superposition of our SARS-CoV-2 S-ACE2 structure with the crystal structure of SARS-CoV RBD-ACE2 (PDB: 2AJF), suggesting that the RBM T470-F490 loop has obvious conformational variations. (C) Binding activities of ACE2-hFc fusion protein to wild-type (wt) and mutant SARS-CoV-2 RBD proteins determined by ELISA. Different structural elements of RBD were colored in the left. Anti-RBD sera and a cross-reactive monoclonal antibody (MAb) 1A10 served as positive controls. Ctr, an irrelevant antibody. The black arrow indicates that mutations in the RBD (RBM-R3) mutant significantly reduced the binding of ACE2-hFc compared with wild-type RBD. (D) Binding of ACE2-hFc fusion protein to wt and single-point mutant forms of SARS-CoV-2 RBD protein measured by ELISA. RBD (Q498A), RBD (V503A), and RBD (Y505A), RBD residues Q498, V503, and Y505 were mutated to Ala, respectively. The downward arrow indicates that the mutation at Y505 completely abolished the binding of ACE2 to RBD protein. OD₄₅₀, optical density at 450 nm.

Fig. 4. Conformational dynamics of the S-ACE2 complex determined by multibody refinement.

(A) Contributions of all eigenvectors to the motion in the S-ACE2 complex, with eigenvectors 1 to 3 dominating the contributions. (B) Top view of the map showing the three swing motions of the first three eigenvectors, with S trimer following the color schema as in Fig. 2, and the two extreme locations of ACE2 illustrated in deep pink and light blue densities. The swing angular range and direction are indicated in dark red arrow. (C) Histograms of the amplitudes along the first three eigenvectors. (D) Atomic models of S-ACE2 and S-closed, colored according to the B-factor distribution [ranging from 100 (blue) to 130 Ų (red)].

Fig. 5. Organization of the resolved N-linked glycans of SARS-CoV-2 S trimer.

(A) Schematic representation of SARS-CoV-2 S glycoprotein. The positions of N-linked glycosylation sequons are shown as branches. A total of 18 N-linked glycans detected in our S-closed cryo-EM map are shown in red, and the remaining undetected ones in black. After ACE2 binding, the glycan density that appears weaker is indicated (*). (B) Surface representation of the glycosylated S trimer in the S-closed state with N-linked glycans shown in red. The location of glycan hole is indicated in black dotted ellipsoid, with the locations of S1/S2 and FP, and glycan at N657 site near the glycan hole indicated. The newly captured glycans at the N17 and N149 sites are indicated in the top view. (C) Surface representation of the glycosylated S-ACE2 complex with N-linked glycans in red.

Fig. 6. The proposed mechanism of ACE2-induced conformational transitions of SARS-CoV-2 S trimer.

Conformational transitions from the closed ground prefusion state (with packed FP, in red) to the transiently open state (step 1) with an untwisting motion (highlighted in dark gray arrow) associated with a downward movement of S1 (red arrow), from the open state to the dynamic ACE2 engaged state (step 2), and then all the way to the refolded postfusion state (step 3). The continuous swing motions of ACE2-RBD within S trimer are indicated by red arrows. The S trimer associated with ACE2 dimer (third panel) was generated by aligning the ACE2 of our S-ACE2 structure with the available full-length ACE2 dimer structure (PDB: 6M1D). The postfusion state was illustrated as a cartoon (PDB: 6XRA).