Extended Data Fig. 7: Site−directed mutagenesis of RBD-62, using the affinity-enhancing mutations.
From: SARS-CoV-2 variant prediction and antiviral drug design are enabled by RBD in vitro evolution
Fifteen mutations were predicted to enhance RBD-ACE2 binding or stability6. Mutation K417T (grey) was introduced from the B6(FA) library. These mutations were evaluated for enhancing the affinity of RBD-62 towards ACE2 and their effect on stability. Single measurements only. (A) Impact of mutations, on top of RBD-62, on ACE2 binding (y-axis) and yeast surface expression. Three mutations (orange circles), which had the highest impact on expression, were combined in RBD-71 (red triangle). (B) Localization of stabilizing (yellow) and binding-enhancing mutations depicted in the RBD structure (PDB ID 6m17, best rotamer is shown). (C) Binding curve of RBD-71 with RBD-62 binding curve fit (in grey) for comparison. All data points, taken in three independent repeats, are shown. (D) Normalized protein melting curves for RBD-WT, RBD-62, RBD-52, and RBD-71 measured using the Tycho NT.6 (NanoTemper).
