Cryoelectron microscopy structures of a human neutralizing antibody bound to MERS-CoV spike glycoprotein

Abstract

Neutralizing monoclonal antibodies (mAbs) against highly pathogenic coronaviruses represent promising candidates for clinical intervention. Here, we isolated a potent neutralizing monoclonal antibody, MERS-S41, from a yeast displayed scFv library using the S protein as a bait. To uncover the neutralization mechanism, we determined structures of MERS-S41 Fab in complex with the trimeric spike glycoprotein by cryoelectron microscopy (cryo-EM). We observed four distinct classes of the complex structure, which showed that the MERS-S41 Fab bound to the "up" receptor binding domain (RBD) with full saturation and also bound to an accessible partially lifted "down" RBD, providing a structural basis for understanding how mAbs bind to trimeric spike glycoproteins. Structure analysis of the epitope and cell surface staining assays demonstrated that virus entry is blocked predominantly by direct competition with the host receptor, dipeptidyl peptidase-4 (DPP4).

Keywords: MERS-CoV; cryo-EM structures; neutralization mechanism; neutralizing antibody; spike glycoprotein.

FIGURE 1

Antibody MERS-S41, isolated from a scFv yeast display library, has potent neutralizing activity. (A) The selection flowchart. (B) Neutralization of the MERS-S41 IgG against pseudotyped MERS-CoV. Antibody 7D10 IgG targets NTD of MERS-CoV S and P2B-2F6 targets RBD of SARS-CoV-2, as positive and negative controls, respectively. (C) Surface plasmon resonance curves showing binding of MERS-CoV spike glycoprotein to immobilized MERS-S41 IgG. Data are shown as different colored lines and the best fit of the data to a 1:1 binding model is shown in black.

FIGURE 2

Cryo-EM structures of the MERS-S41 Fab bound to MERS-CoV spike trimer. (A) Class 1 shows MERS-S41 Fab bound to one “up” RBD. (B) Class 2 shows one “up” RBD bound by two MERS-S41 Fabs. (C) Class 3 shows two “up” RBDs bound by two MERS-S41 Fabs. (D) Class 4 shows two “up” RBDs bound by three MERS-S41 Fabs. In panels (A–D), the MERS-CoV spike trimer NTDs are colored in blue, RBDs in green and other domains in gray. The light chain and heavy chain of MERS-S41 are colored in magenta and purple. The representations are models, shown as surface using Chimera X.

FIGURE 3

The binding interface of the MERS-S41 Fab and MERS-CoV RBD. (A) Structure of the MERS-S41 Fab bound to RBD. The RBD core subdomain is colored in blue, the receptor-binding subdomain in green, the MERS-S41 light chain in magenta, and the MERS-S41 heavy chain in purple. (B) The MERS-S41 epitope. The MERS-CoV RBD is shown in green surface with residues within 4Å of MERS-S41 Fab labeled and colored pink. (C) The MERS-S41 paratope. The MERS-S41 light and heavy chain surfaces are shown in magenta and purple, respectively. Residues within 4Å of the RBD are colored green and labeled in either magenta or purple, depending on whether they are from the light or heavy chain. (D) The DPP4 binding site. The MERS-CoV RBD is shown in green surface with residues within 4Å of DPP4 colored orange and labeled. Residues labeled in pink are also in the MERS-S41 epitope. (E) The RBD/MERS-S41 Fab structure superposed on the RBD-DPP4 structure (PDB 4L72). The MERS-CoV RBD is shown in green surface, DPP4 shown in yellow cartoon, and MERS-S41 Fab in pink cartoon.

FIGURE 4

Breadth of MERS-S41 neutralization. (A) Neutralizing analysis of MERS-S41 IgG against MERS-CoV wild-type (WT) and its variant mutants. Site-directed mutations were introduced into the EMC strain to create 23 variant mutants according to natural mutations of MERS-CoV S. (B) Summary of MERS-S41 IgG mediated inhibition of infection by all pseudotyped viruses. IC₅₀ neutralization titers for mutant EMC S variants are presented relative to wild-type S.