Neutralization of SARS-CoV-2 by Destruction of the Prefusion Spike

Abstract

There are as yet no licensed therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2, initiating conformational changes that drive membrane fusion. We find that the monoclonal antibody CR3022 binds the RBD tightly, neutralizing SARS-CoV-2, and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilizing CR3022 epitope is inaccessible in the prefusion spike, suggesting that CR3022 binding facilitates conversion to the fusion-incompetent post-fusion state. Cryogenic electron microscopy (cryo-EM) analysis confirms that incubation of spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope could be useful therapeutically, possibly in synergy with an antibody that blocks receptor attachment.

Keywords: CR3022; SARS-CoV-2; X-ray crystallography; antibody; cryo-electron microscopy; epitope; neutralization; receptor binding domain; spike; therapeutic.

Graphical abstract

Figure 1

Sequence Alignment between the RBDs of SARS-CoV(-1) and SARS-CoV-2 Residue numbers are those of SARS-CoV-2 RBD. Conserved amino acids have a red background, secondary structures are labeled on the top of the sequence, and the glycosylation site is marked with a blue hexagon. Residues involved in receptor binding are marked with magenta disks. Blue disks mark the residues involved in interactions with the CR3022 heavy chain (Vh), cyan disks mark the residues interacting with the CR3022 light chain (Vl), and green disks mark those with both chains.

Figure 2

Dose-Response Curve for PRNT with CR3022 For CR3022 at a starting concentration of 1.36mg/mL, the dilutions used were from 1:160 to 1:327,680. The probit mid-point is 1:11,966 (95% confidence intervals: 5,297–23,038).

Figure 3

Overall Structure of RBD/CR3022 Complex (A) Ribbon diagram showing the structure of the RBD/CR3022 complex with the RBD shown in gray, CR3022 heavy chain in magenta, and light chain in cyan. The heavy chain CDR1-3 are labeled as H1–H3 and the light chain CDR1-3 as L1–L3 (where visible). (B) Closeup of the antigen-antibody binding interface in cartoon representation. (C) Similar view to (B), but showing the RBD as a surface. (D) The RBD of the RBD/ACE2 complex has been overlapped with the RBD of the RBD/CR3022 complex to show the relative positions of the antigenic and receptor binding sites. ACE2 is drawn as a salmon ribbon.

Figure 4

Surface Properties of SARS-CoV-2 RBD The central panel is a cartoon depiction rainbow colored from blue for the N terminus to red for the C terminus; the view is the same as for (A)–(D). The secondary structure is labeled along with the glycosylated residue N343 (in magenta) and the position of the domain termini (N and C). (A) Surface representation of RBD, with the solvent-accessible area buried by ACE2 receptor binding colored in salmon and that buried by CR3022 (heavy chain in blue and light chain in cyan). (B) Sequence differences shown in red between SARS-CoV and SARS-CoV-2 RBDs, mapped on the surface of SARS-CoV-2 RBD. (C) The surface buried in the pre-fusion conformation of the spike shown in green. (D) The electrostatic surface of SARS-CoV-2 RBD contoured at ± 5 T/e (red, negative; blue, positive).

Figure 5

Details of Contacts between the RBD and CR3022 (A and B) Contacts of the RBD with CR3022 heavy chain CDR1 (H1) and CDR2 (H2) (A), and with CDR3 (H3) (B). (C) Interactions between the RBD and the light chain CDR1 (L1). Main chain backbones are shown as thinner sticks and side chains as thick sticks (RBD, salmon; heavy chain, blue; light chain, cyan). The yellow broken sticks represent hydrogen bonds or salt bridges. (D) Ligplot (Laskowski and Swindells, 2011) representation of the interface details (chain identifiers: L, CR3022 light chain; H, CR3022 heavy chain; E, RBD).

Figure 6

The CR3022 Binding Regions Are Inaccessible in the Pre-fusion Form of the S Protein (A)–(C) An overview. (A) The pre-fusion state of the S protein with all RBDs in the down conformation (generated by superposing our RBD structure on the pre-fusion trimer [Wrapp et al., 2020]). The viral membrane would be at the bottom of the picture. All of S1 and S2 are shown in yellow apart from the RBD, which is shown in gray, with the CR3022 epitope colored green. (B) A cut-way of the trimer showing, in red and the di-peptide (residues 986–987), which has been mutated to PP to inhibit conversion to the post-fusion state. Note the proximity to the CR3022 epitope. (C) A top view of the molecule (also used for [D]–[F]). One of the RBDs has been drawn in light gray in the down configuration and hinged up in dark gray, using the motion about the hinge axis observed for several coronavirus spikes, but extending the motion sufficiently to allow CR3022 to bind. The PP motif is shown in red and the glycosylated residue N343 in magenta. (D–F) The trimer viewed from above. All RBDs down (D), one RBD up (E), and one RBD rotated (F) (as in [C]) to allow access to CR3022. (G–I) Equivalent structures to (D)–(F) but viewed from the side, in (H) bound ACE2 is shown and in (I) CR3022 is shown.

Figure 7

Cryo-EM Reconstructions (A) and (B) are derived from the 50 min incubation, (C) from the 3 h incubation. (A) Cryo-EM map and fitted model of the prefusion spike: left top-view, right side-view. Note RBD I is in the up conformation. (B) Cryo-EM map and fitted model of the dimeric RBD/CR3022 complex, with each monomer labeled A and B. (C) Reconstruction from 3-h incubation dataset to indicate how the CR3022 Fab/RBD complex might be accommodated within one oligomeric unit.