Evolution of the SARS-CoV-2 spike protein in the human host

The SARS-CoV-2 spike glycoprotein is the major surface antigen of the virus. Its function is to bind the host receptor ACE2 and mediate subsequent entry into human host cells. Prior work showed that the pandemic D614G substitution, localized at a monomer–monomer interface of the spike trimer, increases the propensity to adopt an open conformation competent for receptor binding and decreases shedding of S1 from virion spikes, consistent with increased stability of the pre-fusion conformation. Recently emerging variants have acquired additional substitutions in spike, including changes at monomer–monomer interfaces, at the receptor-binding site, and near the furin-cleavage site. Here, the structures and receptor-binding properties of spikes from the B.1.1.7 (Alpha) and B.1.351 (Beta) variants are examined using fusion-stabilized spikes to compare pre-fusion conformations with the original Wuhan strain and a D614G-only variant from previous studies. Two recent studies showed increased RBD accessibility and enhanced receptor binding by Alpha and Beta spikes; this work extends those observations, providing structural insights into receptor binding and increased infectivity of these variants.

The study builds on a body of work describing the effects of the D614G substitution on spike conformation, stability, S1 shedding, and infectivity, as well as reports of additional mutations in emerging variants at the receptor-binding site and furin-cleavage site. Prior studies indicated increased RBD receptivity and enhanced ACE2 binding for Alpha and Beta variants. References cited include structural analyses of coronavirus spikes, characterization of D614G effects, and epidemiological assessments of variant transmissibility and severity.

- Construct design: SARS-CoV-2 spike constructs derived from the Wuhan strain sequence were cloned into pCDNA3.1. Variant spikes were stabilized with the 2P substitutions (K986P, V987P). Furin S1/S2 site was either kept intact (PIHRRAR; 2P constructs) or mutated (FURP constructs with PRSARS). Variant-specific mutations (e.g., D614G, N501Y, K417N, P681H; and others for Alpha, Beta, and mink Y453F) were introduced relative to Wuhan. - Protein expression and purification: Spikes were expressed in suspension Expi293/293F cells at 37°C, 125 rpm, and purified as previously described for D614G spikes. For experiments reducing cleavage, a furin inhibitor (Arginyl-Valyl-Arg chloromethylketone) was added at 100 µM 30 min post-transfection. ACE2 was produced as previously described. - Biolayer interferometry (BLI): ACE2 binding kinetics (kon, koff) were measured to calculate Kd values for variant spikes (see Supplementary Fig. 7). - CryoEM sample prep and data collection: Samples (various spike/ACE2 and spike states for Alpha, Beta, Delta) were vitrified on gold grids (R2/2). Data were collected on Titan Krios microscopes at 300 kV using Falcon 3 cameras in electron-counting mode (calibrated pixel size ~0.82 Å). - Image processing: Motion correction with MotionCor2; CTF estimation with CTFFIND; particle picking and refinement in cryoSPARC with Non-Uniform Refinement and local resolution estimation; maps sharpened with B-factor procedures. - Model building and refinement: Initial models from prior SARS-CoV-2 spike/RBD structures (e.g., D614G PDB IDs 7BNM, 7BNN, 7BNO; Wuhan ACE2-bound PDB ID 7T8S) were fitted. Manual adjustments in COOT; real-space refinement and validation in PHENIX; measurements in Chimera/CCFM. - Experimental variables: Presence/absence of furin cleavage (modulated by sequence or inhibitor), comparison across Wuhan, D614G-only, Alpha, Beta (and mink Y453F) backgrounds; assessment of trimeric vs monomeric states upon ACE2 binding.

- Enhanced ACE2 affinity due to N501Y (dependent on D614G): BLI Kd values (nM): Wuhan (D614) 90.7 ± 14.3; Alpha (G614) 16.7 ± 7.6; Beta (G614) 44.7 ± 10.5; engineered Alpha G614D 93.0 ± 14.0 (similar to Wuhan). The N501Y substitution forms a more stable interaction with ACE2 (e.g., hydrogen bonding with ACE2 Lys353), increasing affinity. - Alpha spike stability and cleavage: Alpha spike remains fully trimeric upon ACE2 binding under conditions where Wuhan and Beta spikes frequently dissociate to monomeric S1–S2/ACE2 complexes. Alpha is almost fully cleaved at S1/S2 due to P681H (more basic furin site), correlating with trimer stability. When expressed with a furin inhibitor (reduced cleavage), >50% of particles were monomeric S1–S2/ACE2 upon ACE2 incubation, indicating cleavage contributes to trimer stability in the ACE2-bound state. - Beta spike adopts open conformations: CryoEM shows Beta spike trimers predominantly in the open state, in contrast to Wuhan (previously ~83% closed under similar conditions). The K417N substitution on the D614G background is implicated in stabilizing the open trimer required for receptor binding. - Differential effects on ACE2 interactions: While N501Y enhances binding in both Alpha and Beta, Beta’s K417N removes a salt bridge between RBD and ACE2, yielding a smaller affinity increase than Alpha. - D614G facilitates RBD accessibility: The D614G substitution increases the proportion of accessible RBD-binding sites, enhancing avidity for host cells and decreasing S1 shedding from virion spikes. - Additional observation: The mink-associated Y453F RBD substitution increases ACE2 binding affinity. - Data resources: Multiple cryo-EM maps deposited (EMD-14225, EMD-14226, etc.) and models in PDB (e.g., 6O25, 7R10, 7R12, 7R13, 7R16, 7R18, 7R19, 7R21).

Findings demonstrate that SARS-CoV-2 variants have evolved distinct but convergent mechanisms to increase effective receptor engagement and likely transmissibility in humans. The early D614G substitution primes spikes for receptor binding by favoring open RBD conformations and stabilizing pre-fusion trimers (reduced S1 shedding). Alpha further enhances ACE2 binding through N501Y and increases S1/S2 cleavage (via P681H), resulting in near-complete cleavage and a trimer that remains intact upon ACE2 binding. This stability may promote multivalent ACE2 engagement and improved cell attachment. Beta also leverages N501Y for increased ACE2 affinity but includes K417N, which disrupts a salt bridge with ACE2, moderating the affinity gain while stabilizing the open trimeric conformation needed for receptor binding. Collectively, these changes increase the accessibility and avidity of RBDs for ACE2 on host cells, which can translate into higher infectivity. The structural and biophysical insights reconcile prior observations of increased transmissibility for these variants and highlight how combinations of substitutions within RBD, inter-protomer interfaces, and cleavage sites tune spike function in the human host.

This study provides structural and biophysical evidence that the Alpha and Beta SARS-CoV-2 variants have evolved spike protein changes that enhance ACE2 binding and favor conformations conducive to infection. N501Y increases receptor affinity (on a D614G background), Alpha’s P681H promotes near-complete S1/S2 cleavage and trimer stability upon ACE2 binding, and Beta’s K417N with D614G stabilizes the open trimer required for receptor engagement. These adaptations likely contribute to increased transmissibility in humans. Future research should investigate how these mechanisms generalize across additional variants (e.g., Delta, Omicron) and in the context of full-length, membrane-embedded spikes and intact virions, including effects on neutralizing antibody recognition and viral fitness in vivo.

- Equilibria of conformational states cannot be robustly quantified from cryo-EM micrographs; proportions reported (e.g., open vs closed) are qualitative under matched conditions. - Use of prefusion-stabilized (2P) soluble spikes and in vitro ACE2 may not fully capture behavior of native, membrane-embedded spikes on intact virions. - Furin cleavage status was manipulated by sequence or inhibitor; residual differences in processing could affect observed stability. - Binding measurements via BLI reflect soluble interactions and may differ from cell-surface avidity effects. - The dataset focuses on Alpha and Beta with comparisons to Wuhan and D614G; generalization to other lineages requires further study.