Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants

The study addresses how emerging SARS-CoV-2 variants alter interactions between the spike receptor-binding domain (RBD) and the human ACE2 receptor, affecting binding affinity and cell entry. By quantifying hACE2 binding, solving RBD–hACE2 complex structures, and testing pseudovirus entry and neutralization by soluble hACE2, the work seeks to elucidate the molecular determinants underlying increased transmissibility and to inform therapeutic strategies.

- Protein production: Cloning and expression of human ACE2 ectodomain (residues 19–615) using a baculovirus expression system with C-terminal His tag; purification by Ni-NTA affinity and further buffer exchange. Production of SARS-CoV-2 RBD proteins (WT and variants: Alpha, Beta, Gamma, Mink-Y453F, Mink-F486L, Mink-N501T; plus single/double mutants such as K417N, K417T, E484K, N501Y/K417N, N501Y/K417T) in mammalian cells with C-terminal His tag; purification by HisTrap affinity chromatography and dialysis. - Binding assays: Flow cytometry (FACS) using BHK cells stably expressing GFP and hACE2 to quantify RBD binding (anti-His APC detection). Surface plasmon resonance (SPR) on Biacore S with CM5 chip to determine equilibrium dissociation constants (KD) for interactions between hACE2 (or mink ACE2) and RBD variants; measurements performed at 25 °C in PBS-based running buffer; analysis over three independent replicates. - Structural biology: Preparation of RBD–hACE2 complexes for Alpha, Beta, Gamma, Mink-Y453F, and Mink-F486L; crystallization by vapor-diffusion sitting drop; data collection at Shanghai Synchrotron (BL17U); data processing with HKL2000; structure determination by molecular replacement in Phenix using known RBD–ACE2 model; refinement to resolutions 2.4–2.9 Å. - Computational analysis: Molecular dynamics (MD) simulations using GROMACS (CHARMM36, TIP3P) and MM/PBSA calculations to estimate binding energies and residue-level contributions, focusing on Mink-Y453F vs WT. - Pseudovirus generation and entry assays: Production of VSV-GFP-based pseudoviruses bearing SARS-CoV-2 spike proteins (D614G, Alpha, Beta, Gamma, Mink-Y453F, Mink-F486L, Mink-N501T; and Beta-N417I); infection of hACE2-positive Huh7 cells; quantification of GFP-positive cells by FACS; statistical analysis by one-way ANOVA with Tukey’s test. - Neutralization by soluble ACE2: Testing soluble hACE2 protein for inhibition of variant pseudovirus entry; reporting EC50 and fold-changes relative to WT across triplicate experiments. - Sequence surveillance: Download and analysis of S gene sequence frequencies for variants from GISAID to contextualize transmissibility trends.

- Increased RBD–hACE2 binding for multiple variants: • FACS binding to hACE2-expressing cells: WT RBD 33.49% positive cells vs Alpha 82.7%, Beta 48.1%, Gamma 57.7%, Mink-Y453F 84.7%, Mink-N501T 76.8%; Mink-F486L showed reduced binding compared to Alpha. • SPR: WT KD ≈ 26.34 nM. Variants displayed stronger binding (lower KD), with approximately 3-fold (Alpha), 5-fold (Beta), 8-fold (Gamma), and 4-fold (Mink-Y453F and Mink-N501T) increases in binding strength vs WT. Mink-F486L bound with ~4-fold lower affinity than WT. - Enhanced RBD binding to mink ACE2 (miACE2): WT RBD bound miACE2 with ~8.16 μM KD (~310-fold weaker than to hACE2). Mink-Y453F, Mink-N501T, and Mink-F486L RBDs showed ~21-, ~9-, and ~15-fold higher affinity to miACE2 than WT, respectively, supporting mink adaptation. - Structural basis of altered affinity (crystal structures at 2.4–2.9 Å): • N501Y strengthens interactions via cation–π with hACE2 K353 and π–π with hACE2 Y41, explaining increased affinity (Alpha, Beta, Gamma). • E484K is distant from the hACE2 interface and has minimal effect on binding. • K417N/T disrupts the K417–D30 salt bridge, slightly reducing affinity relative to N501Y-only backgrounds (Beta/Gamma lower than Alpha). • F486L weakens the hydrophobic interaction with hACE2 Y83, decreasing hACE2 binding but alleviates steric clash with mink ACE2 residue 178, improving mink ACE2 binding. • Y453F (Mink-Y453F) creates more favorable hydrophobic packing near hACE2 H34, increases hydrogen-bonding network (additional H-bond between hACE2 Y83 and RBD Y489), and yields more favorable binding energy by MM/PBSA. - Pseudovirus entry into hACE2-positive Huh7 cells: Alpha, Beta, Mink-N501T, and Mink-Y453F showed increased transduction relative to D614G; Gamma showed similar transduction to D614G; Mink-F486L did not increase entry. Substitutions outside RBD also affect entry (Beta-N417I retained higher entry similar to Beta, exceeding Gamma). - Soluble hACE2 neutralization: Soluble hACE2 efficiently inhibited entry of most variant pseudoviruses, with stronger inhibition for variants showing higher hACE2 affinity (notably Gamma); Mink-F486L was less inhibited.

The findings demonstrate that several emerging SARS-CoV-2 variants enhance binding to hACE2 through defined structural mechanisms, notably N501Y-mediated cation–π and π–π interactions and Y453F-mediated hydrophobic optimization. While K417N/T reduces a key salt bridge, the net effect in Beta and Gamma remains increased affinity due to N501Y. These affinity changes generally translate into higher pseudovirus entry for Alpha, Beta, Mink-N501T, and Mink-Y453F, though Gamma’s entry remained comparable to D614G, indicating that mutations outside the RBD and other steps of entry/fusion modulate infectivity. Enhanced binding of mink-associated mutations (Y453F, F486L, N501T) to mink ACE2 supports cross-species adaptation and explains observed transmission dynamics in minks. The robust neutralization of variant pseudoviruses by soluble hACE2 underscores receptor decoys as a therapeutic strategy that is resilient to immune-escape mutations targeting antibody epitopes. Together, structural, biophysical, and functional data clarify how specific mutations reshape the RBD–ACE2 interface to influence transmissibility and species tropism.

This work provides integrated molecular, structural, and functional insights into how emerging SARS-CoV-2 variants alter RBD–ACE2 interactions. Five variants (Alpha, Beta, Gamma, Mink-Y453F, Mink-N501T) increased hACE2 binding affinity; crystal structures delineate residue-level mechanisms (N501Y, K417N/T, E484K, Y453F, F486L). These changes correlate with increased pseudovirus entry for most variants and suggest adaptations to mink ACE2 for mink-associated mutations. Soluble hACE2 effectively neutralizes variant pseudoviruses, supporting ACE2-based decoys as a broadly acting therapeutic approach. Future work should include live-virus validation across cell types and animal models, comprehensive assessment of mutations outside RBD that impact entry and fusion, and optimization of engineered ACE2 with enhanced affinity and pharmacokinetics.

- Pseudovirus-based assays may not fully recapitulate live-virus entry and replication dynamics. - The contribution of mutations outside the RBD to entry and transmissibility was inferred but not comprehensively dissected. - Sampling bias and detection limitations in sequence surveillance may affect variant frequency interpretations. - Neutralization tests focused on soluble hACE2 against pseudoviruses; broader panels with live viruses and clinical isolates are needed.