Figure 5.

B9-scFv binds to a conserved, cryptic site on the RBD.A, Wood’s plots showing the summed differences in deuterium uptake in SARS-CoV RBD at 2 min of exposure to deuterium, comparing RBD alone to RBD in the presence of B9-scFv. Wood’s plots were generated using Deuteros (46). Peptides colored in blue are protected from exchange in the presence of B9-scFv. Peptides with no significant difference between conditions, determined using a 99% confidence interval (dotted line), are shown in gray (n = 3). B, amino acid sequence alignments of SARS-CoV (Urbani) and SARS-CoV-2 (Wu-1) RBDs (aa 319–591). Orange boxes around the sequence indicate a protected region on the SARS-CoV RBD when incubated with B9-scFv as identified by HDX. A cyan asterisk above the sequence indicates the residues in SARS-CoV-2 that were mutated to their SARS-CoV equivalent in the binding studies shown in (C). C and D, binding of B9-scFv to SARS-CoV-2 (Wu-1) Spike or the SARS-CoV-2 Spike carrying the indicated mutations. Quantification of binding is shown in (C; n = 3); selected FACS plots are shown in (D). For (C), data presented is mean ± SD (n = 3). E, modeling the proposed epitope of B9-scFv onto a crystal structure of SARS-CoV-2 RBD (blue) bound to ACE2 (red) (PDB: 6M0J). HDX protected region 1 (RBD residues 449–467) is in yellow, while residues within 5 Å of this are in green or cyan. Those in cyan indicate those mutations tested in (C and D), arrows indicate the mutations that increase binding. The alignments of the relevant regions of the SARS-CoV and SARS-CoV-2 RBDs are presented beneath the structural models. Mutations that improve binding in (C and D) are highlighted in red and labeled above the sequence. FACS, fluorescence-activated cell sorting, HDX, hydrogen-deuterium exchange; PDB, Protein Data Bank; RBD, receptor-binding domain.