SARS-CoV-2 RBDs with Q498H mutations have high affinity for ACE2.

Abstract

(a) Soluble forms of HuACE2, WH-RBD and mutant RBD were expressed in HEK 293 cells and purified on nickel affinity columns. Purified proteins were resolved by SDS/PAGE and detected by Coomassie staining. Molecular masses are shown in kDa. (b) Biolayer interferometry plots of kinetics of binding RBD to HuACE2. WH-RBD, or the indicated RBD, was in solution and Hu-ACE2 immobilized on sensors. Binding was measured with RBD concentrations of 12, 30, 60, 120nM for WH-RBD and 6, 12, 30, 60nM for the mutants. Fitted curves are in red. Data are shown for single experiments representative of at least three independent experiments for each RBD. (c) Kinetic binding constants for RBD binding to HuACE2 measured using biolayer interferometry. Data are shown as means and SEM for at least three independent experiments. (d) Cellular binding of RBD and S477N/Q498H-RBD. Vero-E6 cells were incubated without RBD (C) or with a range of concentrations of WH-RBD (circles) or S477N/Q498H-RBD (triangles) as indicated, at 37°C for 15 mins before washing, antibody staining of bound RBD with fluorescently conjugated antibody, and flow cytometry. Data are shown as mean fluorescence intensity vs RBD concentration (nM) for a single experiment representative of three.</p