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.