Receptor binding, immune escape, and protein stability direct the natural selection of SARS-CoV-2 variants

Abstract

Emergence of new severe acute respiratory syndrome coronavirus 2 variants has raised concerns related to the effectiveness of vaccines and antibody therapeutics developed against the unmutated wildtype virus. Here, we examined the effect of the 12 most commonly occurring mutations in the receptor-binding domain of the spike protein on its expression, stability, activity, and antibody escape potential. Stability was measured using thermal denaturation, and the activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding to the human angiotensin-converting enzyme 2 and to neutralizing human antibody CC12.1, respectively. Our results show that mutants differ in their expression levels. Of the eight best-expressed mutants, two (N501Y and K417T/E484K/N501Y) showed stronger affinity to angiotensin-converting enzyme 2 compared with the wildtype, whereas four (Y453F, S477N, T478I, and S494P) had similar affinity and two (K417N and E484K) had weaker affinity than the wildtype. Compared with the wildtype, four mutants (K417N, Y453F, N501Y, and K417T/E484K/N501Y) had weaker affinity for the CC12.1 antibody, whereas two (S477N and S494P) had similar affinity, and two (T478I and E484K) had stronger affinity than the wildtype. Mutants also differ in their thermal stability, with the two least stable mutants showing reduced expression. Taken together, these results indicate that multiple factors contribute toward the natural selection of variants, and all these factors need to be considered to understand the evolution of the virus. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that variant.

Keywords: ACE2; COVID-19; SARS-CoV-2; antibodies; protein binding; protein expression; protein function; protein stability; protein structure; variants.

Figure 1

Structures of SARS-CoV-2 RBD (colored blue) interacting with ACE2 and CC12.1 Fab.A, ACE2 (colored gray; PDB ID: 6M0J). B, CC12.1 Fab (colored green; PDB ID: 6XC2). Both showing the most frequently mutating residues in RBD—K417, N439, Y453, S477, T478, E484, S494, and N501 (colored red). RBM is shown in yellow color. The single mutants of RBD used in this study were K417N, N439K, Y453F, S477N, T478I, E484K, S494P, and N501Y (alpha variant). A double mutant (E484K/N501Y) and triple mutants corresponding to beta variant (K417N/E484K/N501Y) and gamma variant (K417T/E484K/N501Y) were also used. The position of Y453 is not visible in the surface view of RBD interacting with CC12.1 Fab as it is buried at the interface. ACE2, angiotensin-converting enzyme 2; PDB, Protein Data Bank; RBD, receptor-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 2

Comparison of relative expression of RBD and its mutants.A, SDS-PAGE showing relative amounts of expressed RBD and its mutants. M represents molecular weight markers (from top to bottom: 180, 130, 100, 70, 55, 35, and 25 kDa). B, relative expression of mutants quantified from the band intensities in SDS-PAGE in panel A. RBD, receptor-binding domain.

Figure 3

Secondary structure characterization of RBD and its mutants.A, purified RBD and its mutants. B, ACE2 and CC12.1 ScFv. M represents molecular weight marker (from top to bottom: 180, 130, 100, 70, 55, 35, 25, 15, and 10 kDa). C, comparison of secondary structures of RBD and its mutants using far-UV CD spectroscopy. Table 1 lists the proportion of various secondary structures when the spectra were deconvoluted using BeStSel software. ACE2, angiotensin-converting enzyme 2; RBD, receptor-binding domain; ScFv, single-chain fragment variable.

Figure 4

Thermal denaturation melts of RBD and its mutants obtained using far-UV CD spectroscopy.A–I show the data for the wildtype RBD, single amino-acid mutations K417N, Y453F, S477N, T478I, E484K, S494P, N501Y, and for the triple mutant K417T/E483K/N501Y, respectively. The solid lines show the fits to a two-state unfolding equation (Equation 1 in the Experimental procedures section). Table 2 lists the T_m (midpoint melting temperature) and the ΔH (enthalpy change at T_m) values of RBD variants. RBD, receptor-binding domain.

Figure 5

Binding of RBD and its variants to ACE2 studied using ITC. Panels A–I show the data for the wildtype RBD, single amino-acid mutations K417N, Y453F, S477N, T478I, E484K, S494P, N501Y, and for the triple mutant K417T/E483K/N501Y, respectively. Top panels show the raw thermograms, and the bottom panels show the fit to the integrated heat curve. Table 3 lists the interaction parameters from the data fit. ACE2, angiotensin-converting enzyme 2; ITC, isothermal titration calorimetry; RBD, receptor-binding domain.

Figure 6

Binding of RBD and its variants to CC12.1 ScFv studied using ITC. Panels A–I show the data for the wildtype RBD, single amino-acid mutations K417N, Y453F, S477N, T478I, E484K, S494P, N501Y, and for the triple mutant K417T/E483K/N501Y, respectively. Top panels show the raw thermograms, and the bottom panels show the fit to the integrated heat curve. Table 4 lists the interaction parameters from the data fit. ITC, isothermal titration calorimetry; RBD, receptor-binding domain; ScFv, single-chain fragment variable.

Figure 7

Structures of SARS-CoV-2 RBD colored blue interacting with ACE2 and CC12.1 Fab.A, ACE2 colored gray and B, CC12.1 Fab colored green, showing the interface residues of RBD in yellow along with the side chains. The frequently mutating residues that are part of binding interface are colored red and also shown with their side chains. ACE2, angiotensin-converting enzyme 2; RBD, receptor-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.