A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV

Abstract

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has now become a pandemic, but there is currently very little understanding of the antigenicity of the virus. We therefore determined the crystal structure of CR3022, a neutralizing antibody previously isolated from a convalescent SARS patient, in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein at 3.1-angstrom resolution. CR3022 targets a highly conserved epitope, distal from the receptor binding site, that enables cross-reactive binding between SARS-CoV-2 and SARS-CoV. Structural modeling further demonstrates that the binding epitope can only be accessed by CR3022 when at least two RBDs on the trimeric S protein are in the "up" conformation and slightly rotated. These results provide molecular insights into antibody recognition of SARS-CoV-2.

Fig. 1. Crystal structure of CR3022 in complex with SARS-CoV-2 RBD.

(A) Overall topology of the SARS-CoV-2 spike glycoprotein. NTD, N-terminal domain; RBD, receptor binding domain; SD1, subdomain 1; SD2, subdomain 2; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane region; IC, intracellular domain; N, N terminus; C, C terminus. (B) Structure of CR3022 Fab in complex with SARS-CoV-2 RBD. CR3022 heavy chain is orange, CR3022 light chain is yellow, and SARS-CoV-2 RBD is light gray. (C and D) Epitope residues on SARS-CoV-2 are shown. CDR loops are labeled. Epitope residues that are conserved between SARS-CoV-2 and SARS-CoV are shown in cyan, and those that are not conserved are shown in green. (D) Epitope residues that are important for binding to CR3022 are labeled. Epitope residues are defined here as residues in SARS-CoV-2 RBD with buried surface area > 0 Ų after Fab CR3022 binding, as calculated with Proteins, Interfaces, Structures and Assemblies (PISA) (34). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; F, Phe; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (E) Several key interactions between CR3022 and SARS-CoV-2 RBD are highlighted. CR3022 heavy chain is orange, CR3022 light chain is yellow, and SARS-CoV-2 RBD is cyan. Hydrogen bonds are represented by dashed lines.

Fig. 2. Conservation of epitope residues.

(A) Sequence alignment of SARS-CoV-2 RBD and SARS-CoV RBD. CR3022 epitope residues are highlighted in cyan. ACE2-binding residues are highlighted in magenta. Nonconserved epitope residues are marked with asterisks. (B to E) Interactions between the nonconserved epitope residues and CR3022 are shown. Amino acid variants observed in SARS-CoV are in parentheses. SARS-CoV-2 RBD is cyan, CR3022 heavy chain is orange, and CR3022 light chain is yellow. Residues are numbered according to their positions on the SARS-CoV-2 S protein sequence. (B) Whereas SARS-CoV-2 has an Ala at residue 372, SARS-CoV has Thr, which introduces an N-glycosylation site at residue N370. (C) The potential location of N370 glycan in SARS-CoV RBD is indicated by the dotted box. CR3022 is shown as an electrostatic potential surface presentation with units of kT/e, where e is the charge of an electron, k is the Boltzmann constant, and T is temperature in kelvin. (D) P384 interacts with T31, S96, and T100 of CR3022 heavy chain. Ala at this position in SARS-CoV would allow the backbone to adopt a different conformation when binding to CR3022. (E) T430 forms a hydrogen bond (dashed line) with S27f of CR3022 light chain. Met at this position in SARS-CoV would instead likely insert its side chain into the hydrophobic pocket formed by Y27d, I28, Y32, and W50 of CR3022 light chain.

Fig. 3. The relative binding location of CR3022 with respect to receptor ACE2 and other SARS-CoV RBD monoclonal antibodies.

(A) Structures of CR3022–SARS-CoV-2 RBD complex and ACE2–SARS-CoV-2 RBD complex (11) are aligned on the basis of the SARS-CoV-2 RBD. ACE2 is green, RBD is light gray, and CR3022 is yellow. (B) Structural superposition of CR3022–SARS-CoV-2 RBD complex, F26G19–SARS-CoV RBD complex [Protein Data Bank (PDB) ID 3BGF] (35), 80R–SARS-CoV RBD complex (PDB ID 2GHW) (36), and m396–SARS-CoV RBD complex (PDB ID 2DD8) (16).

Fig. 4. Model of the binding of CR3022 to the homotrimeric S protein.

(A) RBD in the S proteins of SARS-CoV-2 and SARS-CoV can adopt either an up conformation (blue) or a down conformation (red). PDB ID 6VSB (cryo-EM structure of SARS-CoV-2 S protein) (17) is shown. (B and C) CR3022 epitope (cyan) on the RBD is exposed in (B) the up conformation but not in (C) the down conformation. (D) Binding of CR3022 to single-up conformation would clash (indicated by the red dashed circles) with the S protein. Clash 1: CR3022 variable region (yellow) clashes with the S2 domain. Clash 2: CR3022 constant region (brown) clashes with NTD. Clash 3: CR3022 variable region clashes with the neighboring RBD that is in the down conformation. (E) Clash 3 is resolved when the neighboring RBD is in the up conformation (i.e., S protein in double-up conformation). (F) All clashes are resolved if the targeted RBD is slightly rotated in the double-up conformation. The curved arrow indicates the change in CR3022 orientation due to the slight rotation of the ­RBD. Of note, given that the elbow angle between the constant and variable domains of CR3022 is the same as observed in our crystal structure, our model shows that a maximum rotation angle of ~45° for the RBD would avoid all clashes. However, the elbow region of an antibody is known to be highly flexible. Therefore, the rotation angle of the RBD could be much smaller when the spike trimer is bound to CR3022. (G) Binding of CR3022 immunoglobulin G (IgG) and m396 IgG to recombinant RBD proteins from SARS-CoV-2 and SARS-CoV (left panel) and to SARS-CoV-2 and SARS-CoV viruses (right panel). Black lines indicate mean ± standard deviation of three technical replicates. OD₄₅₀, optical density at 450-nm wavelength.