Structural Definition of a Unique Neutralization Epitope on the Receptor-Binding Domain of MERS-CoV Spike Glycoprotein

Abstract

The major mechanism of antibody-mediated neutralization of the Middle East respiratory syndrome coronavirus (MERS-CoV) involves competition with the cellular receptor dipeptidyl peptidase 4 (DPP4) for binding to the receptor-binding domain (RBD) of the spike (S) glycoprotein. Here, we report a unique epitope and unusual neutralizing mechanism of the isolated human antibody MERS-4. Structurally, MERS-4 approached the RBD from the outside of the RBD-DPP4 binding interface. Such binding resulted in the folding of the β5-β6 loop toward a shallow groove on the RBD interface critical for accommodating DPP4. The key residues for binding are identified through site-directed mutagenesis. Structural modeling revealed that MERS-4 binds to RBD only in the "up" position in the S trimer. Furthermore, MERS-4 demonstrated synergy with several reported antibodies. These results indicate that MERS-4 neutralizes MERS-CoV by indirect rather than direct competition with DPP4. This mechanism provides a valuable addition for the combined use of antibodies against MERS-CoV infection.

Keywords: Middle East respiratory syndrome; antibody epitope; coronavirus; crystal structure; neutralizing antibody.

Graphical abstract

Figure 1

Crystal Structures of MERS-CoV RBD in Complex with MERS-4 and Its Variant MERS-4V2 (A) Overall structures of the RBD/MERS-4 Fab and the RBD/MERS-4V2 scFv (right) complexes. The RBD core subdomain was in blue, while the receptor-binding subdomain was in green, the MERS-4 light chain in magenta, the MERS-4 heavy chain in cyan, the MERS-4V2 VL in orange, and the MERS-4V2 VH in red. (B) Structural superimposition of the RBD/MERS-4 and the RBD/MERS-4V2 complexes and schematic illustration of the MERS-CoV RBD (right). (C and D) MERS-4 (C) and MERS-4V2 (D) interact with the β5-β6, β6-β7, and β7-β8 loops of the RBD. See also Figure S1 and Table S1.

Figure 2

The Binding Interface between MERS-CoV RBD and MERS-4V2 (A) Overall view of the interface showing the MERS-4V2 epitope consisting of residues from the β5-β6, β6-β7, and β7-β8 loops of the RBD. (B) Interactions between the RBD residues from the β5-β6 loop, the β7-β8 loop, and MERS-4V2 heavy chain. (C–E) Interactions between the RBD residues from the β6-β7 loop (C and D), β7-β8 loop (E), and the corresponding residues of MERS-4V2 light chain. See also Figure S2 and Table S2.

Figure 3

Comparisons of the DPP4 Binding Site with the Epitopes of MERS-4 and MERS-4V2, and Conformational Change of the RBD β5-β6 Loop in the Antibody-Bound State (A) Structural superimposition showing that the epitopes of MERS-4 and MERS-4V2 (right) are distinct from the DPP4 binding site. A significant conformational difference was found in the RBD β5-β6 loop between antibody-bound and DPP4-bound states. (B) Zoom-in view of the aligned RBD β5-β6 loops in unbound (4KQZ: blue; 4L3N: magenta; 4ZPW: wheat) and DPP4-bound (4L72: cyan; 4KR0: yellow) with either the MERS-4-bound (green) or MERS-4V2-bound (green) (right) states. (C) Patch 2 of the RBD/DPP4 binding interface in which residues Leu506, Asp510, and Glu513 from the RBD β5-β6 loop are critical for DPP4 binding. (D) The steric clashes in the red circle between the β5-β6 loop of the RBD and the DPP4 receptor upon antibody binding. See also Figure S3.

Figure 4

Structural Superimpositions of the RBD/DPP4, RBD/MERS-4, and RBD/MERS-27 Crystal Structures onto the MERS-CoV S Trimer Glycoprotein in Receptor-Binding Inactivated and Activated States (A) MERS-CoV S trimer in receptor-binding inactivated state with all three RBDs in the “down” positions (PDB: 5w9j). The S trimer is shown with semi-transparent surface, in which one S protomer (S1 subunit in green and S2 subunit in orange) is shown as a cartoon. (B–D) Structural superimpositions of the RBD/DPP4 (B), RBD/MERS-4 (C), and RBD/MERS-27 (D) crystal structures onto the S trimer in receptor-binding inactivated state. DPP4 and MERS-4 Fab have steric clashes with the RBD and NTD of the neighboring S protomer, respectively. (E) MERS-CoV S trimer in receptor-binding activated state with one RBD in the “up” positions (PDB: 5w9h). (F–H) Structural superimpositions of the RBD/DPP4 (F), RBD/MERS-4 (G), and RBD/MERS-27 (H) crystal structures onto the S trimer in receptor-binding activated state. The epitope is exposed and readily accessible for binding.

Figure 5

Impact of Mutations in the RBD on Binding and Neutralization Sensitivity to MERS-4 and MERS-4V2 (A) Binding affinities of the wild-type RBD and its mutants (L507A, S508A, L545A, S546A, P547A, and E549A) to MERS-4 and MERS-4V2. (B) Neutralizing activity of MERS-4 against MERS-CoV pseudotyped with wild-type or mutant S glycoprotein (L507A, L545A, S546A, and P547A). The fold changes in IC₅₀ of mutant viruses relative to the wild-type (WT) (>1: increase resistance and <1: increase sensitivity) (right). (C) MERS-4 staining of HEK293T cells expressing wild-type or mutant S glycoprotein. The fold changes in MFI of mutant viruses relative to the wild-type were listed in the table. MFI, median fluorescence intensity. See also Figure S4.

Figure 6

Combination of MERS-4 and MERS-27 in Binding to the RBD Comparison of the experimental SAXS data (black dots) with the theoretical scattering curve calculated from the full-atomic RBD/MERS-4/MERS-27 monomer model (red line) and the theoretical scattering curve calculated from an ensemble consisting of 31% monomer model, 18% dimer model of the RBD/MERS-4/MERS-27, and 51% RBD/MERS-27 (green line). Residuals calculated as I (q) experimental/I (q) model are shown below the scattering curves. See also Figure S6.

Figure 7

Effects of MERS-4 Combined with m336, 5F9, and MERS-27, Respectively, in Neutralizing Pseudotyped MERS-CoV (A) Effects of MERS-4 combined with m336 in neutralizing pseudotyped MERS-CoV. Percent neutralization was calculated for serial 3-fold dilutions of each antibody alone and in combination at constant ratios in a range of concentrations from 27 times to 1/81 of IC₅₀s. The constant ratios of the combined antibodies were their IC₅₀s. On the x axis, a dose of 1 was at the IC₅₀ concentration. Fractional effect (FA) plots generated by the CompuSyn program for MERS-4, m336, and their combination showing dosage versus effect. Median effect plot of calculated CI values (logarithmic) versus FA values, in which a log CI of <0 is synergism and a log CI of >0 is antagonism. Data shown are average values from four independent experiments. (B and C) The percent neutralization, fractional effect, and CI values for MERS-4 combined with 5F9 (B) and MERS-4 combined with MERS-27 (C) were calculated and generated using the same method. See also Figures S5 and S7.