The emergence of SARS-CoV-2 variants of concern (VOCs) poses a significant global health challenge. Some VOCs have demonstrated increased transmissibility and are associated with surges in COVID-19 cases. Understanding the molecular mechanisms underlying the interaction between these VOCs and the human angiotensin-converting enzyme 2 (hACE2) receptor is crucial for developing effective countermeasures. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is the primary determinant of viral entry into host cells by binding to hACE2. Mutations within the RBD can alter its binding affinity for hACE2, potentially impacting viral infectivity and transmission. This study aimed to investigate the molecular basis of hACE2 binding by several emerging VOCs, including Alpha, Beta, Gamma, and three mink-derived variants (Mink-Y453F, Mink-F486L, and Mink-N501T). The goal was to identify key mutations influencing hACE2 binding affinity and to explore the potential for developing therapeutic strategies targeting these interactions. The research is important because it provides critical information that can inform the development of new treatments and preventive measures against the evolving SARS-CoV-2 virus and future pandemics.

Previous studies have shown a strong correlation between RBD mutations and the increased transmissibility and virulence of SARS-CoV-2 variants. Early research established the fundamental role of the RBD in binding to hACE2, and several studies have analyzed the effects of specific mutations on this interaction. Cryo-electron microscopy and X-ray crystallography have been instrumental in visualizing the structure of the spike protein and its interaction with hACE2, providing crucial insights into the molecular mechanisms involved. However, a comprehensive understanding of the molecular basis of hACE2 binding for many emerging VOCs remained lacking prior to this study. The existing literature highlighted the need for detailed analysis of the binding characteristics of multiple variants to hACE2 and the investigation of potential therapeutic interventions.

This study employed a multi-faceted approach combining various experimental techniques to characterize the interaction between SARS-CoV-2 VOC RBDs and hACE2. **1. Binding Affinity Assays:** Flow cytometry (FACS) was used to assess the binding affinity of different RBDs (wild-type and variants) to hACE2-expressing cells. Surface plasmon resonance (SPR) provided quantitative measurements of the equilibrium dissociation constant (KD) for each RBD-hACE2 interaction. **2. Crystallography:** X-ray crystallography was used to determine high-resolution structures of five variant RBD-hACE2 complexes (Alpha, Beta, Gamma, Mink-Y453F, and Mink-F486L). These structures provided detailed insights into the molecular interactions between the RBDs and hACE2. **3. Pseudovirus Transduction Assays:** To investigate the functional consequences of altered RBD-hACE2 binding, pseudoviruses incorporating the spike protein of each variant were generated and used to infect hACE2-expressing cells. The transduction efficiency was measured using flow cytometry to quantify the entry of pseudoviruses into cells. **4. Neutralization Assays:** The neutralization potential of soluble hACE2 protein against SARS-CoV-2 variant pseudoviruses was evaluated to assess its ability to block viral entry. **5. Molecular Dynamics (MD) Simulations and Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) Calculations:** MD simulations were performed to study the dynamics of the RBD-hACE2 interactions and to assess the binding free energies using MM/PBSA calculations. This provided complementary information to the experimental results. **6. Gene Cloning, Expression, and Protein Purification:** Standard molecular biology techniques were utilized to clone, express, and purify hACE2 and the variant RBD proteins in appropriate expression systems (e.g., Baculovirus system for hACE2, mammalian expression system for RBDs). Affinity chromatography was employed for protein purification, ensuring high purity for subsequent experiments. **7. Complex Preparation and Crystallization:** Purified hACE2 and RBD proteins were mixed, and the complexes were crystallized using a vapor-diffusion sitting-drop method. High-quality crystals were obtained and used for X-ray diffraction data collection. **8. Data Collection and Structure Determination:** X-ray diffraction data were collected at a synchrotron facility. Structure determination involved data processing, phasing, model building, and refinement. **9. Statistical Analysis:** Statistical significance of the results was determined using appropriate methods such as one-way ANOVA with Tukey's multiple comparison test.

The study revealed that five out of six SARS-CoV-2 variants examined (Alpha, Beta, Gamma, Mink-Y453F, and Mink-N501T) exhibited increased binding affinity for hACE2 compared to the wild-type virus. This increased affinity was confirmed by both FACS and SPR assays. The crystal structures revealed specific residues involved in the enhanced binding. For instance, the N501Y mutation in Alpha, Beta, and Gamma variants introduced new favorable noncovalent interactions (cation-π and π-π stacking) with hACE2, thereby increasing binding strength. The K417N and K417T mutations in Beta and Gamma variants, respectively, disrupted a salt bridge with hACE2, contributing to the higher affinity. The Mink-Y453F mutation introduced a hydrophobic interaction with hACE2, increasing binding energy and the number of hydrogen bonds. In contrast, the Mink-F486L mutation showed decreased binding affinity to hACE2. Consistent with the binding affinity changes, pseudovirus transduction assays showed increased entry efficiency for Alpha, Beta, Mink-N501T, and Mink-Y453F pseudoviruses compared to the D614G strain. Gamma pseudovirus showed similar transduction efficiency to D614G, suggesting that mutations outside the RBD may also play a role in cell entry. The neutralization assays demonstrated that soluble hACE2 protein effectively inhibited entry of most variant pseudoviruses, highlighting its potential as a therapeutic agent. Notably, the three mink-origin variants displayed higher binding affinity for mink ACE2, providing a molecular explanation for their efficient transmission among minks. Analysis of SARS-CoV-2 variant S sequences from the GISAID database showed that the frequencies of Alpha, Beta, Mink-Y453F, and Mink-N501T increased rapidly, suggesting their adaptation to human transmission. Conversely, Mink-F486L, which had reduced affinity for hACE2, did not efficiently transmit to humans. The study also highlights that factors beyond RBD-hACE2 binding may influence the transmissibility of SARS-CoV-2 variants.

The findings of this study provide valuable insights into the molecular mechanisms underlying the enhanced binding of several SARS-CoV-2 VOCs to hACE2. The identification of key residues involved in these interactions has implications for the development of targeted therapeutics. The demonstrated efficacy of soluble hACE2 in preventing viral entry highlights its potential as a therapeutic agent against these variants. The differential binding affinities and transduction efficiencies observed between variants emphasize the complex interplay between mutations in the RBD and other factors affecting viral entry and transmission. The study also underscores the importance of monitoring the emergence and spread of SARS-CoV-2 variants and understanding their adaptation to different hosts. Future research should investigate the impact of other mutations beyond the RBD on viral transmission and explore the development of modified hACE2-based therapies with enhanced binding affinities to emerging variants.

This research provides a detailed molecular understanding of how recent SARS-CoV-2 variants interact with the hACE2 receptor, revealing increased binding affinities for several variants. This knowledge is critical for developing effective antiviral therapies, particularly soluble hACE2-based treatments. Future research should focus on characterizing newly emerging variants and investigating potential synergies with other therapeutic approaches to combat the ongoing COVID-19 pandemic.

The study primarily focused on in vitro experiments using pseudoviruses, which may not fully recapitulate the complexity of in vivo infection. The neutralization assays employed pseudoviruses, which may not perfectly reflect the behavior of authentic viruses. The limited number of variants analyzed may not encompass the full diversity of circulating strains. Further research with authentic viruses and in vivo models is needed to validate the findings and assess the full clinical implications.