Structural model and binding kinetics of SARS-CoV-2 RBD variants to ACE2
To address the questions of antibody binding strength and competition mechanistically, we have expressed the RBD of the isolate P.1 exhibiting RBD mutations (K417N, E484K and N501Y) (RBDTrip), two of which are located in the RBM (E484K, N501Y) (shown in Fig. 1A)8. In addition, we generated each of the respective single mutants (RBDK417N, RBDE484K, and RBDN501Y). All RBDs were purified to homogeneity and the affinity to recombinant ACE2 was determined by Biolayer Interferometry using Octet technology 9. The BLI assays showed that the affinity of ACE2 for RBDTRIP (shown in Fig. 2B, 2F, Table 1, KD = 10.3 nM) was about twice as high as for RBDWT (shown in Fig. 2A, 2F, Table 1, KD= 20.5 nM). Having in mind that the affinity of the of SARS-CoV-2 for ACE2 is only 4-fold higher compared to SARS-CoV-1, this factor of 2 is expected to be biologically significant and most likely reflects enhanced infectivity. In contrast, the mutation at position 484 in RBD484 did not affect receptor affinity (shown in Fig. 2D, F). For comparison, the dissociation constant observed for RBDN501Y was 3-fold lower (KD = 6.2 nM, shown in Fig. 2C, 2F, Table 1). Interestingly, the mutation at position 417 in RBD417 resulted in completely altered binding properties (shown in Fig. 2E). RBDK417N showed much lower association rates and plateau levels a non-monovalent pattern of dissociation rates (shown in Fig. 2E, Table 1 KD could not be determined in a meaningful way). Presence of aggregates was not responsible for this effect, as purification by size exclusion immediately before measurements did not alter the binding kinetics observed (data not shown). However, since this mutation does not occur on its own in viral strains, we did not further investigate this effect.