Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies

Abstract

The recently reported "UK variant" (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments.

Fig 1. Structure of the SARS-CoV-2 N501Y mutant spike protein ectodomain bound to the ACE2 ectodomain.

(A) Density map for the overall complex at the end of global structure refinement. The 3 spike protein protomers are colored in cyan, purple, and yellow, with the density for the strongly and weakly bound ACE2 proteins in pale red and green, respectively. (B) Improved density map at the contact zone between the receptor binding domain (RBD) and the strongly bound ACE2 protein ectodomain. (C) Visualization of density at the contact zone for Y501 in the RBD and residues Y41 and K353 in ACE2. (D). Ribbon diagram with superposition of the unmutated and N501Y RBD–ACE2 complex (PDB ID 7KMB). (E) Zoomed-in view of the interface, showing a superposition of the structures of unmutated and N501Y mutant spike proteins in complex with ACE2. The carbon atoms of residues in the N501Y mutant and ACE2 in our structure are colored in cyan and pale red, respectively, while those in the structure of the complex between unmutated spike protein and ACE2 are in light gray.

Fig 2. Analysis of V_H Fc ab8 and IgG ab1 interactions with N501Y and unmutated spike.

(A) ELISA analysis of antibody interactions with either N501Y or unmutated spike ectodomain. (B) N501Y or unmutated SARS-CoV-2 S pseudotyped virus neutralization by either V_H Fc ab8 or IgG ab1. (C and D) ELISA analysis of N501Y or unmutated SARS-CoV-2 spike ectodomain binding by soluble ACE2-mFc in the presence of serial dilutions of either (C) IgG ab1 or (D) V_H Fc ab8. ELISA experiments were done at least in duplicate while neutralization experiments were performed twice at least in duplicate, and the average values are shown. Error bars denote the standard error of the mean (SEM).

Fig 3. Structure of V_H ab8 bound to the N501Y mutant spike protein trimer.

(A) Atomic model for the structure of the complex of V_H ab8 (blue) with the N501Y mutant spike protein ectodomain (gray). The structure has 2 receptor binding domains (RBDs) in the down position with well-resolved densities for the bound V_H ab8. The third RBD is in the up position. (B) Cryo-electron microscopy density map after local refinement with fitted coordinates for the contact zone between the RBD and V_H ab8. (C) Density map in the region near 501 for the N501Y mutant spike protein ectodomain showing density for residues Q498, Y501, and Y505. (D and E) Close-up views of the contact zone between the RBD region and ACE2 highlighting residues involved.

Fig 4. Structure of Fab ab1 bound to the N501Y mutant spike protein trimer.

(A and B) Atomic models for the 2 predominant conformations of the spike protein (gray) observed with Fab ab1 (blue) bound to either 2 (A) or 3 (B) receptor binding domains (RBDs) in the up position. (C) Cryo-electron microscopy density map after local refinement with fitted coordinates for the contact zone between the RBD and Fab ab1. (D) Density map in the region near 501 for the N501Y mutant spike protein ectodomain showing density for residues Q498, Y501, and Y505 in the spike protein and a loop in Fab ab1 that includes S30, the residue closest to Y501. (E and F) Close-up views of the contact zone between the RBD region and ACE2 highlighting residues involved.

Fig 5. Comparison of the structures of complexes formed by the spike protein ectodomain with the ACE2 ectodomain, V_H ab8, and Fab ab1.

(A–C) Open-face views of the receptor binding domain (RBD) from the vantage point of ACE2 (A), V_H ab8 (B), and Fab ab1 (C), with the residues involved in contact shaded in red, yellow, and blue, respectively. (D–F) Space-filling model view of ACE2 (D), V_H ab8 (E), and Fab ab1 (F) in contact with the RBD (structure shown in ribbon format). (G) Superposition of the structures of the complex of the RBD with ACE2, V_H ab8, and Fab ab1 to show their relative footprints on the RBD surface.