Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation

Abstract

The outbreak of a novel betacoronavirus (2019-nCov) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for urgently needed vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure (MCM) development we determined a 3.5 Å-resolution cryo-EM structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also show biophysical and structural evidence that the 2019-nCoV S binds ACE2 with higher affinity than SARS-CoV S. Additionally we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to nCoV-2019 S, suggesting antibody cross-reactivity may be limited between the two virus RBDs. The atomic-resolution structure of 2019-nCoV S should enable rapid development and evaluation of MCMs to address the ongoing public health crisis.

Figure 1.. Structure of 2019-nCoV S in the prefusion conformation.

(A) Schematic of 2019-nCoV S primary structure, colored by domain. Domains that were excluded from the ectodomain expression construct or could not be visualized in the final map are colored white. SS= signal sequence, NTD= N-terminal domain, RBD= receptor-binding domain, SD1= subdomain 1, SD2= subdomain 2, S1/S2= S1/S2 protease cleavage site, S2′= S2′ protease cleavage site, FP= fusion peptide, HR1= heptad repeat 1, CH= central helix, CD= connector domain, HR2= heptad repeat 2, TM= transmembrane domain, CT= cytoplasmic tail. Arrows denote protease cleavage sites. (B) Select 2D class averages of the particles that were used to calculate the 2019-nCoV S reconstruction (left). Side and top views of the prefusion structure of the 2019-nCoV S protein with a single RBD in the “up” conformation (right). The two RBD “down” protomers are shown as cryo-EM density in either white or gray and the RBD “up” protomer is shown in ribbons, colored corresponding to the schematic in Fig 1A.

Figure 2.. Structural comparison between 2019-nCoV S and SARS-CoV S.

(A) A single RBD “down” monomer of 2019-nCoV S is shown in ribbons, colored according to Figure 1. A monomer of SARS-CoV S is also shown in ribbons, colored white (PDB ID: 6CRZ). (B) The following structural domains from 2019-nCoV S have been aligned to their counterparts from SARS-CoV S; NTD (top left), RBD (top right), SD1 and SD2, (bottom left) and S2 (bottom right).

Figure 3.. 2019-nCoV S binds human ACE2 with high affinity.

(A) SPR sensorgram showing the binding kinetics for human ACE2 and immobilized 2019-nCoV S. Data are shown as black lines and the best fit of the data to a 1:1 binding model is shown in red. (B) Negative-stain EM 2D class averages of 2019-nCoV S bound by ACE2. Averages have been rotated so that ACE2 is positioned above the 2019-nCoV S protein with respect to the viral membrane. A cartoon depicting the ACE2-bound 2019-nCoV S protein is shown (right) with ACE2 in blue and S protein monomers colored tan, pink and green.

Figure 4.. Antigenicity of the 2019-nCoV RBD.

(A) The SARS-CoV RBD is shown as a white molecular surface (PDB ID: 2AJF), with residues that vary in the 2019-nCoV RBD colored red. The ACE2 binding site is outlined with a black dotted line. (B) A biolayer interferometry sensorgram that shows binding to ACE2 by the 2019-nCoV RBD-SD1. Binding data are shown as a black line and the best fit of the data to a 1:1 binding model is shown in red. (C) Biolayer interferometry to measure cross-reactivity of the SARS-CoV RBD-directed antibodies S230, m396 and 80R. Sensortips with immobilized antibodies were dipped into wells containing 2019-nCoV RBD-SD1 and the resulting data are shown as a black line.