Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding

Abstract

The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor and is a major determinant of host range and a dominant target of neutralizing antibodies. Here, we experimentally measure how all amino acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD’s surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity-enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open analysis pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations observed during viral surveillance.

Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms

“…Surveillance efforts have led to the identification of a number of S mutants among circulating SARS-CoV-2 isolates. Several naturally occurring RBD mutations were shown to abrogate interactions with known mAbs and to reduce immune sera binding, raising concerns that viral neutralization escape mutants could emerge or be selected under pressure from mAbbased anti-viral treatments (42). To investigate if S2E12-and S2M11-mediated neutralization might be affected by SARS-CoV-2 polymorphism, we tested binding of either mAb to 29 S protein variants (corresponding to mutations detected in circulating SARS-CoV-2 isolates) expressed at the surface of CHO cells.…”

A human monoclonal antibody targeting a conserved pocket in the SARS-CoV-2 receptor-binding domain core

“…E) Relative surface binding of 47D11, (F) ACE2, (G) CR3022 and (H) an anti-FLAG antibody to full-length SARS2 spike epitope mutants, determined by fluorescence-activated cell sorting. I) Antibody-mediated neutralization of infection of luciferase-encoding VSV particles pseudotyped with wild-type, V367A or V367F SARS2-S. J) Surface representation of the 47D11 bound SARS2-S RBD coloured according to mean mutation effect on expression (red indicates more constrained)( 45 ). The Fab is shown as a ribbon diagram.…”

“…Total cellular expression of mutants was comparable to wildtype spike protein as demonstrated by an antibody targeting the C-terminal appended Flag-tag on the spike proteins (Figure 3H), suggesting that mutations in the RBD hydrophobic core have a detrimental effect on protein folding, compromising the tertiary structure of the RBD. A recent study reported deep mutational scanning of SARS2-S RBD residues, revealing how mutation of each of the RBD residues affects expression of folded protein and its affinity for ACE2( 45 ). When the mean mutation effect on expression was mapped on the 47D11 bound RBD, we observed that the hydrophobic pocket, targeted by 47D11, is highly mutationally constrained (Figure 3J).…”

“…Volume measurements were performed using CASTp 3.0, using a probe radius of 1.2 Å( 65 ). In order to colour the 47D11 bound RBD surface according to each residues mean mutational effect on expression, the pdb file was populated with the mean mutation effect on expression values described by Starr et al( 45 ). The UCSF Chimera ‘MatchMaker’ tool was used to obtain RMSD values, using default settings.…”

Attenuated influenza virions expressing the SARS-CoV-2 receptor-binding domain induce neutralizing antibodies in mice

“…The ∆NA(RBD) construct, described in Fig 1A and the first results subsection below, used the protein sequence for the receptor binding domain (RBD) of Spike from SARS-CoV-2 (isolate Wuhan-Hu-1, Genbank accession number MN908947, residues 331-531). The nucleotide sequence was codonoptimized as previously described [28]. To facilitate surface expression of RBD, the murine immunoglobulin H chain V-region leader sequence was added to the N-terminus.…”