Structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80R

Abstract

Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease that caused pandemic spread in 2003. The etiological agent of SARS is a novel coronavirus (SARS-CoV). The coronaviral surface spike protein S is a type I transmembrane glycoprotein that mediates initial host binding via the cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as the subsequent membrane fusion events required for cell entry. Here we report the crystal structure of the S1 receptor binding domain (RBD) in complex with a neutralizing antibody, 80R, at 2.3 A resolution, as well as the structure of the uncomplexed S1 RBD at 2.2 A resolution. We show that the 80R-binding epitope on the S1 RBD overlaps very closely with the ACE2-binding site, providing a rationale for the strong binding and broad neutralizing ability of the antibody. We provide a structural basis for the differential effects of certain mutations in the spike protein on 80R versus ACE2 binding, including escape mutants, which should facilitate the design of immunotherapeutics to treat a future SARS outbreak. We further show that the RBD of S1 forms dimers via an extensive interface that is disrupted in receptor- and antibody-bound crystal structures, and we propose a role for the dimer in virus stability and infectivity.

FIGURE 1

Diffraction patterns of complex crystal. The complex crystals display a lattice-translocation defect caused by translocations in the crystal packing between neighboring layers along the a* direction. a, a* is nearly vertical, in the plane of the paper, and the defect results in periodic sharp-diffuse-diffuse rows of diffraction intensities (the bottom left quadrant is a zoom-in of the boxed area). b, a* is nearly parallel to the x-ray beam and perpendicular to the paper, and the defect is not evident.

FIGURE 2

h layer intensities before and after correction.a, the lattice defect results in a strong-weak-weak pattern of intensities along h, which were corrected (b) according to the procedure of Wang et al. (10).

FIGURE 3

Stereo 2F_o - F_c electron density map of the S1-RBD-80R complex at the S1-80R interface. S1 and 80R residues are shown in red and blue, respectively, with selected residues labeled. Contour level = 1.5σ.

FIGURE 4

Structure of the S1-RBD-80R complex.a, overall structure of the complex. Antibody variable region light chain is in blue, and heavy chain is in magenta; S1-RBD is in red. b, comparison between the S1 RBD-80R complex (red and yellow) and the S1 RBD-ACE2 complex (blue and green) overlaid on the S1-RBD domain. c, close-up of the interface. Selected S1 side chains are in red; 80R is in blue; hydrogen bonds are in cyan. CDRs (L1-L3 and H1-H3) and the framework (FW) loop (interacting with the extended loop of S1) are labeled. There is an aromatic ring stacking between Tyr⁴⁸⁴ (S1) and Tyr¹⁰² (80R). Tyr⁴⁸⁴ and Tyr¹⁰² are in turn coordinated by hydrogen bonds between Tyr⁴⁸⁶ (S1) and Tyr¹⁰² (80R) and Tyr⁵³ (80R), and Tyr⁴⁸⁴ (S1) and Tyr⁴³⁶ (S1), respectively. Another intermolecular hydrogen bond occurs between Leu⁴⁷⁸ (S1) and Ser¹⁶³ (80R). Asn¹⁶⁴ (80R) makes intramolecular hydrogen bonds with Arg²²³ (80R). Intramolecular hydrogen bonding between Tyr¹⁰³ (80R) and Asp¹⁸² (80R) may be important for maintaining the 80R structure at the interface and may be important for S1 RBD-80R binding. Cys⁴⁷⁴ (S1), Ala⁴⁷¹ (S1), and Ser¹⁹⁷ (80R) form another intermolecular hydrogen bonds that may stabilize the S1 RBD-80R interface.

FIGURE 5

Stereo comparison of the S1-RBD domain. Uncomplexed (dimeric) S1-RBD is in red, complex with 80R antibody is in green; complex with ACE2 is in blue. Helical elements A and B and the C terminus are labeled. The receptor-binding surface, including the extended loop, is highly conserved in all three structures, lies at the back of the field, and is not visible in this view. Root mean square deviation values for pairwise comparisons are 0.9-1.1 Å for main chain residues excluding helix A (residues 350-360), helix B (370-381), and N and C termini before residue 323 or after residue 502. The small differences in these regions of the complexed S1-RBDs presumably arise from the different crystal environments. The large changes in the uncomplexed S1-RBD are a consequence of dimer formation.

FIGURE 6

Structure of the S1-RBD dimer.a, S1 monomers are in red and blue, related by a vertical 2-fold axis. The receptor binding surfaces and C termini are indicated. b, same as in a but rotated by 90° about a horizontal axis to show the molecular dyad. c, hypothetical model of the S1-RBD dimer with two molecules of ACE2 bound, showing steric overlap (circled). The view is rotated about a vertical axis compared with a in order to minimize the overlap in projection. Two Fab fragments can bind the dimer without steric hindrance (not shown). A full-length dimeric antibody could presumably cross-link neighboring dimers on the viral surface, but the geometry is inappropriate for binding both sites on a single dimer simultaneously.