Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity

Binding of SARS-CoV-2 S protein variants to ACE2 receptor and antibodies

We used the previously described S-GSAS-D614G S ectodomain as a template here (Fig. 1 and table S1) (16) (referred to as “D614G spike” hereafter). This template includes SARS-CoV-2 S residues 1 to 1208, an Arg-Arg-Ala-Arg (RRAR) to Gly-Ser-Ala-Ser (GSAS) substitution that renders the furin cleavage site inactive, and a foldon trimerization motif at the spike C terminus, followed by a C-terminal TwinStrep tag. All purified S proteins showed similar migration profiles upon SDS–polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography (SEC), with high-quality spike preparations confirmed by negative-stain electron microscopy (NSEM) (fig. S1) (32).

We used surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA) to measure spike binding to the ACE2 receptor ectodomain and to Abs (Fig. 1, figs. S2 to S4, and table S2). Abs included RBD-directed, potent nAbs DH1041 and DH1043, whose epitopes overlap with the ACE2 binding site; RBD-directed highly cross-reactive nAb DH1047, which neutralizes SARS-CoV-1, SARS-CoV-2, and bat CoVs; NTD-directed nAbs DH1050.1 and DH1050.2, which bind an antigenic supersite; NTD-directed non-neutralizing Ab (nnAb) DH1052; fusion peptide–directed cross-reactive Ab DH1058; and S2 glycan cluster–directed nnAb 2G12 (fig. S4) (9, 33–37). All variants bound ACE2 at higher levels relative to the D614G spike (Fig. 1C and figs. S2 and S3), with S-GSAS-B.1.1.7 (“B.1.1.7 spike”) displaying the greatest increase. DH1047 showed similar binding levels to all spike variants (Fig. 1D and figs. S2 and S3), consistent with neutralization of B.1.1.7 and B.1.351 by DH1047 (34). The RBD-directed nAb DH1041 showed similar binding levels to the B.1.1.7 and D614G spikes, consistent with its neutralization of the B.1.1.7 pseudovirus (38). The S-GSAS-D614G-K417-E484K-N501Y (the “triple mutant spike”) showed reduced binding to RBD-directed nAbs DH1041 and DH1043. These results are consistent with the inability of class 2 RBD–binding Abs, where the E484K substitution occurs within the epitope, to neutralize variants that harbor the E484K substitution (2).

We tested several variants in the B.1.351 spike backbone (Fig. 1, figs. S2 and S3, and table S1). We found that the commonly occurring 242–244 deletion, and a rare Arg²⁴⁶ → Ile substitution that is included in some reagent panels and candidate vaccines (39), can each affect binding of not only NTD-directed Abs, but also RBD-directed Abs DH1041 and DH1043. Whereas binding of NTD-directed nAbs DH1050.1 and DH1050.2 to B.1.1.7 and B.1.351 spikes was markedly reduced, their binding to the triple mutant spike and S-GSAS-P.1 (or “P.1-like spike”) remained unchanged. This is consistent with neutralization data, where mAbs 5-24 and 4-8 (which target the same antigenic supersite as DH1050.1) lost activity against B.1.351 but neutralized P.1 (40).

In summary, our binding data are consistent with biological data obtained in in vitro neutralization assays, thus establishing that our SARS-CoV-2 S ectodomain constructs are an effective mimic of native spikes and supporting their use for studying structural changes due to amino acid substitutions in spike variants.

Structural analysis of mink-associated “cluster 5” spike mutations

Spillover of SARS-CoV-2 from humans to minks, and then from minks to humans, was first reported in April 2020 in the Netherlands and subsequently independently reported in Denmark, Spain, Italy, the United States, Sweden, and Greece (25). Five S mutations were observed in a variant named “cluster 5”; these included a His⁶⁹/Val⁷⁰ NTD deletion (ΔH69/V70), RBD Tyr⁴⁵³ → Phe (Y453F) substitution, SD2 Ile⁶⁹² → Val (I692V) substitution, and Met¹²²⁹ → Ile (M1229I) in the TM. To understand how these affect spike conformations, we determined cryo-EM structures of S-GSAS-D614G-ΔFV (“ΔFV spike”), which included all but the TM M1229I substitution (Fig. 1, A and B, and table S1). We identified four 3-RBD-down populations, which we named 3D-1, 3D-2, 3D-3, and 3D-4 (PDB 7LWL, 7LWI, 7LWK, and 7LWJ, respectively) (Fig. 2A), refined to overall resolutions of 2.8 to 3.2 Å; three 1-RBD-up populations, which we named 1U-1, 1U-2, and 1U-3 (PDB 7LWM, 7LWN, and 7LWO, respectively), refined to resolutions of 2.8 to 2.9 Å; and one 2-RBD-up population (2U; PDB 7LWP) refined to 3.0 Å (Fig. 2B, figs. S5 and S6, and table S3). A previously unobserved state (M1; PDB 7LWQ, 3.2 Å) was identified, with two RBDs in the down position and no density visible for the entire S1 subunit of the third protomer (Fig. 2C, figs. S5 and S6, and table S3). The 3-RBD-down states were ~43% of the total population, with the rest of the particles constituting “open” states, including ~47% 1-RBD-up, ~7.5% 2-RBD-up, and ~2.3% of the M1 spike. Thus, we observed a modest decrease in the 3-RBD-down state from ~56% that we had reported for the D614G spike, and the appearance of open states (2-RBD-up and M1) that were not observed for the S-GSAS-D614G dataset (16).

Upon closer examination, we noted unusual variability in the S2 subunit of the ΔFV 3-RBD-down structures. We compared these structures either by aligning them using S2 residues 908 to 1035 of the HR1-CH region (fig. S7A) or by calculating difference distance matrices (DDMs) for superposition-free comparisons between pairs of structures (fig. S7, B and C, and supplementary text) (41). Both methods revealed considerable variability in S2, which was most pronounced for the 3D-4 structure (Fig. 2A and fig. S7). By contrast, the three 1-RBD-up structures showed little variability in S2, which suggests that cluster 5 mutations largely affect the 3-RBD-down state (fig. S8) (16). The variation in the S2 region was unexpected because the S2 subunit had appeared relatively invariant in prior studies (16, 42, 43).

We next sought to understand the effect of each amino acid substitution on the functional and structural properties of the spike. The ΔFV spike bound ACE2 with improved affinity over the D614G spike by a factor of ~3.5, resulting from a decreased off-rate mediated by the Y453F substitution (Fig. 2D, fig. S9, and table S2). Although neither the I692V substitution nor ΔH69/V70 affected ACE2 binding affinity, ΔH69/V70 contributed to increased affinity for the NTD-directed nAbs DH1050.1 and DH1050.2. The I692V substitution occurs in SD2, where small changes can translate to large movements in the NTD and RBD regions (Fig. 1) (16, 19). In the D614G spike, Ile⁶⁹² contacts Pro⁶⁰⁰; loss of the methyl due to the I692V substitution increases the distance between Pro⁶⁰⁰ and Val⁶⁹² (fig. S10). We observed disorder in the 3D-4 cryo-EM map, accompanied by the largest separation between Pro⁶⁰⁰ and Val⁶⁹² of all the ΔFV spike 3-RBD-down structures. This local destabilization around the I692V substitution in 3D-4, together with DDM comparisons and superpositions that showed 3D-4 to be the most asymmetric of the 3-RBD-down structures as well as the most variable in the S2 subunit, suggested a role for the I692V substitution in the 3-RBD-down state disorder.

To define and quantify changes in ΔFV spike domain orientations, and to determine how local changes around the SD2 I692V substitution propagate to adjacent domains, we examined its quaternary structure using a vector representation (19). This was accomplished by assigning a central coordinate to each domain and calculating angles, dihedrals, and distances between different structural elements (Fig. 2E and supplementary text). Principal components analysis (PCA) of these intraprotomer vector relationships showed that the 3D-4 protomers occupied a distinct cluster (Fig. 2F), consistent with the DDM analysis (fig. S7, B and C). The two RBD-down protomers in M1(A and C) were similar to 3D-1(A), 3D-2(C), 1U-1(A), and 2U(C) protomers along the first principal component (PC1), with M1(A) separating from M1(C) in PC2 into a 3D-1(A)–containing cluster. Both 3D-1(A) and 3D-3(C) occupied extreme positions in the vector set for angles involving the NTD′, subdomains, and the RBD that mimic the 1U-1(A) structure (fig. S11). Because constraints on RBD-down protomers are relaxed in spikes with at least one RBD in the up position, this may represent a particularly stable protomer position. Together, the vector clustering is consistent with structural observations for the 3D-4 structure and indicates that loss of a single S1 protomer in M1 allowed its two other RBD-down protomers to relax to a configuration resembling RBD-down protomers in 1-RBD-up spikes.

We next examined the angle formed by the NTD′, SD2, and SD1 domain centers, termed θ₃, and a dihedral describing how the NTD′, SD2, SD1, and RBD rotate relative to one another, termed ϕ₃ (Fig. 2, E to H). The 3D-4 protomers occupied a distinct ϕ₃ and θ₃ angle cluster (fig. S12); in particular, the 3D-4(A) protomer ϕ₃ dihedral differed markedly from the primary cluster in the direction of up-state protomers (Fig. 2H, inset). Consistent with the PCA clustering, the θ₃ angles of 3D-1(A), 1U-1(A), and 2U(C) were similar to those of the M1 protomers. The 3D-2(C), 3D-1(A), and 1U-1(A) protomers displayed ϕ₃ dihedrals similar to those of the M1 protomers (Fig. 2, G and H). The similarity of the M1 protomers and the up-state protomers suggests that the M1 state occurs to release strain from 3-RBD-down configurations induced by the cluster 5 mutations. Comparing the 3D-4(A) S1 subunit structure to that of M1(A) demonstrated the marked differences in their RBD positioning, whereas alignment of M1(A) S1 subunit to 1U-1(A) showed their similarity (Fig. 2I).

Comparing the ΔFV spike 3-RBD-down structures to our previously published D614G spike structures (PDB 7KE4, 7KE6, 7KE7, and 7KE8) revealed that the 3D-1 and 3D-2 protomers closely matched 7KE4 and 7KE8, respectively, in their intraprotomer ϕ₃ and θ₃ angles (fig. S11, A and B). Two protomers in the 3D-4(B and C) structure resembled two protomers in the 7KE8(A and B) D614G spike structure in their ϕ₃ dihedrals. Both the 7KE8 and 3D-4 structures displayed marked asymmetry, with the third protomer in each occupying an extreme dihedral angle; in 7KE8(C), the NTD and RBD are rotated toward S2, whereas 3D-4(A) showed a rotation in the opposite direction (fig. S11C). As a result of contact between SD1 and NTD′, this results in global shifts of S1 elements away from S2. These shifts, together with close contact between S2 and these S1 domains, result in changes in S2 structure leading to the variability observed in our structural analysis (fig. S7). The large separation of S1 from S2 in the 3D-4(A) protomer (fig. S11C) suggests that it could be an intermediate that leads to the S1-dislocated M1(B) state. The 3D-3 structure also lacked a close match (fig. S11, A and B). Alignment of 3D-3 to its most similar D614G down-state trimer structure, 7KE7, indicated similar but less extreme differences in domains, which suggests that 3D-3 is yet another intermediate structure leading to the pre-M1 3D-4 state. Thus, by combining cryo-EM classifications and vector analysis, we tracked the origin of the observed instability in the ΔFV spike and found evidence of instability in two 3-RBD-down structures (3D-3 and 3D-4) that leads to dislocation of a S1 protomer in M1.

In summary, our data show that interspecies adaptation involves improved receptor binding affinity of the ΔFV spike mediated primarily by the RBD Y453F substitution. The observed increase in RBD-up states may also contribute to higher levels of ACE2 binding by providing more receptor-accessible sites. We found no evidence in the binding data for immune evasion at the dominant neutralization sites; this is consistent with previous findings that neutralization potency of a panel of RBD antibodies was not notably affected by Y453F or ΔH69/V70 (38). Structural analysis revealed destabilization of the 3-RBD-down state and loss of tight regulation of its conformation in the mink-associated ΔFV spike. We can infer from these structures that in the virion-associated spike these changes could have an impact on spike stability, possibly leading to premature S1 shedding.

Structural analysis of the SARS-CoV-2 S protein B.1.1.7 variant

The B.1.1.7 variant emerged in the UK in September 2020 and spread worldwide, with reports of increased transmissibility, virulence, and mortality (44). An RBD N501Y substitution results in improved ACE2 affinity (45). The N501Y substitution, either on its own or in combination with the NTD ΔH69/V70 deletion or the SD2 P681H mutation, does not substantially affect serum neutralization elicited by current vaccines (1, 38, 46, 47). Although susceptible to RBD-directed nAbs such as DH1041, DH1043, and DH1047 (9, 38), B.1.1.7 shows increased resistance to NTD-directed Abs including 4A8 (PDB 7C2L), 5-24, and 4-8 (10, 48). This resistance was attributed to the ΔY144 deletion, which occurs in a NTD loop that forms an antigenic supersite (49) also targeted by the DH1050.1 nAb (PDB 7LCN) (50).

Our binding data were consistent with published neutralization data (Fig. 1, Fig. 3A, and figs. S2 and S3). B.1.1.7 spike affinity for ACE2 was improved over the D614G spike by a factor of ~5 as a result of the N501Y substitution. We measured nanomolar affinity of the B.1.1.7 spike for NTD-directed nAb DH1050.1, albeit at substantially reduced binding levels relative to the D614G spike (Fig. 3A, figs. S2, S3, and S9, and table S2), consistent with impairment of the NTD antigenic supersite in B.1.1.7 (49), while retaining robust binding to most RBD-directed antibodies.

To visualize the impact of the amino acid variations on the spike conformation, we determined cryo-EM structures of the B.1.1.7 spike (Fig. 3, B to D, figs. S13 and S14, and table S3). Multiple populations of the 3-RBD-down and RBD-up states were identified, with a higher proportion of RBD-up particles observed for the B.1.1.7 spike (~1.8:1 RBD-up/RBD-down) relative to the D614G spike (~0.8:1) (16) and the mink-associated ΔFV spike (~1.3:1) (Fig. 2, A to C). Three populations of 3-RBD-down spike were refined to 3.2 to 3.6 Å (Fig. 3B, figs. S13 and S14, and table S3), each showing visible asymmetry with weaker density for one of its RBDs (Fig. 3B), suggestive of enhanced mobility. We identified several RBD-up structures, including a typical 1-RBD-up state (Fig. 3C) and two 1-RBD-up populations with the up RBD and its adjacent NTD disordered (Fig. 3D). We identified states with 2- or 3-RBD up (fig. S13G) that were not detected in the D614G spike (16). We were unable to obtain high-resolution reconstructions of these populations because of their limited particle numbers and preferred orientations of the particles. Unlike the mink-associated ΔFV spike structures, DDM analysis of the B.1.1.7 structures did not show variability in S2 (fig. S15). The apparent increase in RBD mobility in the B.1.1.7 spike 3-RBD-down structures suggested a reduced barrier for up-state transition due to weakening of down-state contacts. RBDs in their down state contact an adjacent NTD and another RBD via interprotomer protein-protein and protein-glycan contacts (Fig. 3B, inset) (51, 52). Transition from the down to the up state replaces these contacts with differing RBD-to-NTD and RBD-to-RBD contacts (Fig. 3C, inset).

We next sought to understand how variations that are distal from the RBD/NTD region influence the B.1.1.7 spike conformational distribution. These variations spanned multiple domains including SD1 [Ala⁵⁷⁰ → Asp (A570D)], SD2 [Pro⁶⁸¹ → His (P681H)], HR1 [Ser⁹⁸² → Ala (S982A)], CD [Asp¹¹¹⁸ → His (D1118H)], and the linker between SD2 and fusion peptide [Thr⁷¹⁶ → Ile (T716I)] (Fig. 1A). The P681H substitution located near the furin cleavage site could not be visualized because of the disorder in the cryo-EM map in that region. The D1118H substitution, on the other hand, was well resolved and formed a symmetric histidine triad near the base of the spike (Fig. 3E and fig. S14, B and C). Although the histidines were positioned too far from each other for direct hydrogen bonding, water-mediated interactions are feasible at this separation. Moreover, the cryo-EM reconstructions showed evidence for alternate conformations that could place the histidines into closer proximity (fig. S14B). By contrast, the T716I substitution abrogated an intraprotomer hydrogen bond (H-bond) between the Thr⁷¹⁶ side-chain and Gln¹⁰⁷¹ main-chain carbonyls (Fig. 3F), suggesting a local destabilizing effect.

The A570D and S982A substitutions (Fig. 4, A to E), in the SD1 and HR1 regions, respectively, appeared to be counterposing. The A570D substitution resulted in an interprotomer H-bond with the Asn⁸⁵⁶ side chain, reinforcing the stacking of the SD1 loop against the HR1 helix of the adjacent protomer (Fig. 4, A and B). The HR1 S982A substitution, on the other hand, resulted in the loss of an interprotomer H-bond between the Ser⁹⁸² and Thr⁵⁴⁷ side chains (Fig. 4, C and D). Comparing the down (PDB 7KDK) and up (PDB 7KDL) protomers in the D614G spike (16) showed concerted ~5- to 6-Å shifts in the Ala⁵⁷⁰ and Thr⁵⁴⁷ loop positions, with the Thr⁵⁴⁷ loop in the up protomer shifted farther away from Ser⁹⁸² and no longer within H-bonding distance of it (Fig. 4D). Thus, the S982A mutation appears to disable a latch that modulates the RBD up/down equilibrium, thereby increasing RBD up propensity (Fig. 4E). We had previously engineered a construct, named u1S2q, where modulation of a latch involving the Ala⁵⁷⁰ loop was implicated in shifting its RBD up/down equilibrium (19).

To gain insight into how the Ser⁹⁸²-Thr⁵⁴⁷ interprotomer latch affects the spike quaternary structure, we defined a new set of interprotomer vectors (Fig. 4F). Within each S protomer we defined a “unit” comprising the SD1/RBD region and the NTD/NTD′ region of the adjacent protomer with which it interacts. These units are in conformational communication (“Com”) through RBD-to-RBD contacts at the apex, as well as through the SD2 subdomain. We examined the relative disposition of the three units and of SD2 by using a vector network spanning the trimer. For each structure, the protomer that contained the disordered RBD (termed Protomer_B) showed a marked increase in the intraprotomer angle formed by the NTD′, SD2, and an SD2 anchor (SD2a) point (θ_5′) relative to this angle in the other two protomers (Protomer_A and Protomer_C). This occurred in conjunction with a shift in the angle between the SD2, SD2a, and SD1 (θ_6′; Fig. 4G). These angular changes were accompanied by a rotation of SD1 and SD2a about an axis connecting the NTDʹ and SD2 (ϕ_8′) as well as a compensatory rotation of the SD1 to adjacent NTDʹ (ϕ_1′). This compensatory shift occurs as a result of differences in the Ala⁵⁷⁰ loop positions. With the SD2 orientation relative to S2 largely similar to that of the other protomers, these movements can be ascribed to the S982A- and A570D-induced movements of SD1. Together, these changes resulted in disengagement of the NTD from the adjacent RBD, explaining the increase in RBD disorder. Thus, the S982A and A570D pairing acts as an allosteric switch through coupled domain movements.

In summary, structural analysis of the B.1.1.7 spike highlights how allosteric effects of variations in distal regions alter RBD disposition. In B.1.1.7, amino acid substitutions that destabilize the 3-RBD-down or closed state to favor RBD-up or open states are balanced by substitutions that stabilize the prefusion spike conformation. Thus, whereas the T716I substitution disrupts an intraprotomer H-bond, the D1118H histidine triad appears to play a stabilizing role. Similarly, whereas the S982A substitution abrogates an H-bond, facilitating RBD-up movement, the A570D substitution adds an H-bond with Asn⁸⁵⁶, stabilizing interactions between HR1 and SD1. The accumulation of stabilizing contacts in the B.1.1.7 spike, even as it acquires mutations that enable increased presentation of receptor-accessible RBD-up states, may contribute to stabilizing the prefusion spike to prevent premature S1 shedding.

Structural analysis of variants bearing the K417N, E484K, and N501Y RBD mutations

Multiple variants that originated independently in different geographical locations show three amino acid substitutions (K417N, E484K, and N501Y) in the RBD, which suggests convergent evolution and selective advantage of these substitutions. Of these, the E484K mutation is of particular concern because of its location within nAb epitopes, and it has been shown to reduce or eliminate binding to many potent RBD-directed nAbs (2). The E484K and K417N-E484K-N501Y (“triple mutant RBD”) substitutions abolished binding of the potent class 2 RBD nAbs DH1041 and DH1043 to an RBD construct (Fig. 5A and fig. S16) (33). We found, however, that high-affinity binding of DH1041 and DH1043 to S-GSAS-D614G-E484K (“E484K spike”) and S-GSAS-D614G-K417N-E484K-N501Y (“triple mutant spike”) was retained, albeit at reduced levels (Fig. 5, B and C, and figs. S2, S3, and S9).

To understand why some binding to DH1041 and DH1043 was retained for the E484K variant in the context of a S ectodomain, whereas binding was completely abrogated in the RBD-only construct, we studied the effect of the mutations on RBD conformation using molecular dynamics (MD) simulations to compare the native RBD and the triple mutant RBD (model included residues 327 to 529 in each). We built Markov state models of transitions between conformational states from large ensembles of short MD simulations for both constructs (figs. S17 to S20 and table S4; total simulation time ~260 μs each). The Markov models were characterized by a hook-like folded RBD tip (the “Hook” state), which resembled the conformation observed in x-ray crystal structures (33, 53), and a highly dynamic “Disordered” state in which the RBD tip cycles between a variety of conformations (Fig. 5, D and E, and figs. S18 and S19, C and E). Whereas the native RBD displayed a nearly even proportion of Hook versus Disordered states (Fig. 5D), the triple mutant RBD showed a marked increase in the Disordered state (Fig. 5E). These population differences result from an increased transition rate to the Disordered state from the Hook state combined with a slower transition rate back to the Hook state in the triple mutant RBD relative to the native RBD (figs. S18F and S19F). Monitoring the interactions between the residue 484 side chains in each model indicated that the native Glu⁴⁸⁴ hydrogen bonding with the Phe⁴⁹⁰ backbone in particular acted to stabilize the Hook state (fig. S20). In the Disordered state, the Lys⁴⁸⁴ side chain forms fewer interactions across the RBD relative to Glu⁴⁸⁴ (fig. S20B). Together, these results are consistent with the loss in binding of Abs DH1041 and DH1043 to the RBD E484K variant and indicate that the E484K substitution destabilizes the native conformation of the RBD tip, hindering binding of class 2 RBD–directed SARS-CoV-2–neutralizing Abs.

To visualize the impact of RBD tip conformational variability on the spike, we determined cryo-EM structures of the triple mutant spike (Fig. 6A, figs. S21 and S22, and table S3). We identified 3-RBD-down, 1-RBD-up, and 2-RBD-up states, as well as intermediate states that showed one RBD in the up position and another RBD partially up. 3-RBD-down states accounted for ~12% of the total spike population and showed considerable disorder in their RBDs, with the disorder being most pronounced for one of the three RBDs and its contacting NTD (Fig. 6A).

We next studied spikes that, in addition to the RBD K417N-E484K-N501Y substitutions, also contained multiple residue changes in the NTD, and an Ala⁷⁰¹ → Val substitution, found in B.1.351 (Fig. 1, Fig. 6, B and C, and figs. S2, S3, S23, and S24). Despite no additional RBD mutations, binding to RBD-directed nAbs was further reduced (Figs. 1D and 6C), showing that amino acid changes outside the RBD have an allosteric effect on the binding of RBD-directed Abs. A cryo-EM dataset of a B.1.351 spike (Fig. 1) revealed a ~6:1 ratio of RBD-up to 3-RBD-down structures (Fig. 6B). A “consensus” 3-RBD-down state with 212,753 particles was refined to 3.7 Å and displayed remarkably weak RBD density in one of the three RBDs that also appeared detached from its interprotomer-contacting NTD (Fig. 6A, PDB 7LYM). Taken together, these data implicate the K417N-E484K-N501Y substitutions in the RBD disorder observed in the 3-RBD-down states and suggest that the E484K-induced conformational disorder in the RBD tip Hook structure may be the source of the increased RBD-up spike populations due to weakened RBD-to-RBD coupling. In the spike, interprotomer interactions made by the RBD in its up state, and secondary contacts that the bound antibody makes with adjacent RBDs, may play a role in stabilizing antibody binding to the E484K mutant (54), thereby explaining the retention of high-affinity binding, albeit at lower levels.

We next asked whether the weakened RBD-RBD and RBD-NTD coupling involving the disordered RBD had an impact on spike quaternary structure. Domain interface mutations are limited to the RBD in the triple mutant and B.1.351 spike variants (Fig. 1A). Asymmetry in the S1 subunit was observed when aligning the SD2 subdomain of each protomer (Fig. 6D). Patterns in the interprotomer vector network indicated that the triple mutant and B.1.351 spikes were similar in their protomer-to-protomer relationships (Fig. 6E). The absolute positions, however, displayed marked differences (Fig. 6, D and E, and fig. S25), suggesting that the additional mutations in the B.1.351 spike play a role in further modulating spike conformation. Comparing the interprotomer vector networks of these structures with the 3-RBD-down D614G spike structures indicated that the B.1.351 structure was most similar to the D614G 7KE8 structure, whereas the triple mutant spike lacked similarity to any of the D614G structures (fig. S25). This shift toward a more D614G-like state in B.1.351 may indicate the selection of stabilizing mutations to balance the RBD-destabilizing mutations. Together, these results show that amino acid variations in the RBD alone can have marked impacts on S1 quaternary structure, and accumulation of additional variations outside the RBD may in turn modulate RBD conformational changes.

Comparing SARS-CoV-2 variant S ectodomain quaternary structure

The structural results presented here indicate that the primary consequence of conformational adjustments in the SARS-CoV-2 variants is increased propensity for RBD exposure. Our data implicate destabilization of the 3-RBD-down state and involvement of a disordered RBD in this conformational difference. To compare the different approaches that the variants take toward this destabilization, we examined the interprotomer network of each variant spike (Fig. 4F), together with a new RBD-to-RBD and RBD-to-NTD network (Fig. 7A). It is necessary to define a primary protomer for these comparisons because of the asymmetric nature of the spike. We selected the protomer containing the RBD most distant from its adjacent NTD, often the disordered RBD protomer, for this analysis [this protomer is here designated Protomer_A″; a double prime (″) designation was used for all vector measures and domain/protomer names to signify this change]. We also included in our analysis an asymmetric 3-RBD-down reconstruction of our engineered u1S2q S ectodomain (19), and four of our previously published 3-RBD-down D614G spike reconstructions (16). We first examined PCA clustering to identify structurally similar sets (Fig. 7, A and B). The triple mutant and B.1.351 spike structures, as well as ΔFV 3D-1 and 3D-2, clustered with D614G spike structures; the B.1.1.7 and ΔFV 3D-3 structures clustered with u1S2q; and ΔFV 3D-4 differed markedly from all others. The separation of the structures into D614G-like and u1S2q-like is consistent with differing RBD destabilization strategies in the variants that harbor the RBD triple mutants relative to the B.1.1.7 and ΔFV spikes. Examination of the primary vectors reporting on the differences observed in these clusters indicated that the typically disordered RBD protomer, Protomer_A″, is the driver of differences between the two clusters. Positioning of SD1 relative to SD2, defined by the angle θ_4″, in Protomer_A″, and the S2-to-SD2 and S2-to-NTD′ distances, were each indicators of these differences (Fig. 7, B and C).

The interconnected spike domain network suggests that changes in local quaternary arrangements are likely to induce rearrangements in distant domains (Fig. 7D). We therefore examined correlations in quaternary arrangements of SD2, SD2a, SD1, and NTD′ in the full dataset. The variant discriminating SD2-to-SD1 angle θ_4″ (defined in Fig. 4F) displays a considerable number of correlations with quaternary arrangements throughout the network (Fig. 7, E and F, and figs. S26 to S30). This includes correlation with the Protomer_B″ and Protomer_C″ SD2-to-SD1 angles θ_2″ and θ_6″. Correlation was also observed with the interprotomer dihedral angle that defined the rotation of SD2 about axes connecting SD1 and NTD′ from Protomer_B″ to Protomer_C″ ϕ_1″ and Protomer_C″ to Protomer_A″ (ϕ_4″) (Fig. 7, E and F). These, and correlation with dihedral rotation of Protomer_B″ SD2 and Protomer _C″ NTD′ about an axis connecting the SD2 anchor and SD1, ϕ_9″, are mirrored by the Protomer_B″ SD2-to-SD1 angle, θ_6″. The relationships identified show that changes in domain arrangement in one protomer have predictable impacts on the domain arrangements of the other protomers. In the D614G cluster, quaternary arrangements give rise to the marked distance between the disordered RBD and the NTD′ (Fig. 7D). For the triple mutant and B.1.351 spike structures, the RBD tip disorder presumably reduces the stability of its contact with the adjacent RBD, increasing its up-state propensity. Unlike the D614G cluster, in the u1S2q cluster RBDs are all distant from their adjacent NTD (Fig. 7D). Examination of the structures indicated that rearrangements occurred in the orientation of SD1 relative to SD2 and S2. The engineered u1S2q contains mutations only in S2 and in the SD1 Ala⁵⁷⁰ loop that is adjacent to S2. These together increase the up-state population. It is therefore likely that amino acid substitution in SD2 and S2/SD1 in the ΔFV and B.1.1.7 spikes, respectively, are responsible for the increased RBD-up populations in these spikes. Thus, several mechanisms exist by which changes induced in domain interaction strength by spike amino acid substitutions modify RBD positioning.