The emergence of SARS-CoV-2 variants of concern (VoCs) poses a significant challenge to global efforts to control the COVID-19 pandemic. These variants exhibit reduced susceptibility to both infection- and vaccine-induced immunity, necessitating a deeper understanding of their molecular mechanisms. While much has been learned since the emergence of SARS-CoV-2, most mechanistic knowledge is based on early prototype isolates. The receptor-binding domain (RBD) of the spike (S) protein is critical for viral entry and a primary target for neutralizing antibodies. Mutations within the RBD can alter binding affinity to the host cell receptor, angiotensin-converting enzyme 2 (ACE2), and affect antibody recognition. This study aims to quantify the effects of mutations in several VoCs (Alpha, Beta, Gamma, and Kappa) on RBD-ACE2 interaction kinetics, thermodynamics, and structure, ultimately clarifying the impact on viral infectivity and antibody neutralization. The researchers leverage atomic force microscopy (AFM) and molecular dynamics (MD) simulations to achieve this.

Previous research has established the high affinity of the RBD-ACE2 interaction (approximately 10 nM) and the role of multivalent interactions at the interface. AFM has been successfully used to map interaction forces between RBD and ACE2. Computational studies have also investigated the energetic and structural aspects of RBD-ACE2 binding. However, a comprehensive understanding of how specific mutations in VoCs affect these properties at the atomic level is lacking. This study aims to fill this gap by combining experimental and computational methods.

The researchers used a combination of experimental and computational techniques. For the experimental component, they employed atomic force microscopy (AFM) based single-molecule force spectroscopy (SMFS) to measure the binding forces and kinetics between ACE2 receptors immobilized on a surface and RBD proteins from different SARS-CoV-2 variants (Alpha, Beta, Gamma, and Kappa) attached to AFM tips. The binding frequencies and the dissociation constants (Kd) were determined to quantify binding affinity. Dynamic force spectroscopy (DFS) was used to extract kinetic on and off rates. Furthermore, antibody neutralization assays were conducted using monoclonal antibodies (mAbs) against the wild-type RBD to assess the impact of the mutations on antibody binding. For the computational part, all-atom molecular dynamics (MD) simulations of RBD-ACE2 complexes were performed for the wild-type and the VoCs to analyze the energetic and structural changes associated with the mutations. The total energy, Lennard-Jones energy, and electrostatic interactions were calculated and correlated with observed experimental results. The MD simulations provided information on the stability and the nature of intermolecular contacts between the RBD and ACE2.

The AFM experiments revealed that all VoCs exhibited higher binding frequencies and affinities for ACE2 compared to the wild-type RBD. The Gamma and Kappa variants showed the most significant increase in binding affinity. The calculated equilibrium dissociation constants (Kd) reflected this trend: Gamma (Kd = 21 ± 16 nM) < Kappa (Kd = 31 nM) < Beta (Kd = 48.9 ± 1 nM) < Alpha (Kd = 121.8 ± 1 nM) < Delta (Kd = 134.1 nM). MD simulations showed that the increased stability in the RBD-ACE2 complex for several VoCs is mainly due to a gain in van der Waals interactions. This gain of interaction energy is particularly pronounced for the Gamma and Kappa variants. Specific mutations, such as N501Y, were found to significantly contribute to the increased stability. Notably, the N501Y mutation creates additional contacts between the RBD and ACE2, enhancing binding affinity. The E484K mutation affected the binding energetic due to long-range Coulomb interactions. Analysis of antibody neutralization showed that one mAb effectively neutralized all variants, while another lost its neutralization capacity against Beta, Gamma, and Kappa variants, likely due to the presence of the E484 mutation in these variants. The Alpha variant's neutralization was not significantly affected by the N501Y mutation.

The findings demonstrate that the observed increase in transmissibility of SARS-CoV-2 variants of concern can be, at least partially, attributed to increased binding affinity and stability of the RBD-ACE2 complex. The mutations in the VoCs, particularly N501Y and E484K, contribute to this enhanced stability by increasing van der Waals interactions and altering electrostatic interactions, respectively. The increased stability of the RBD-ACE2 interface could explain the escape from neutralization by certain antibodies. The differential neutralization efficacy of the two tested mAbs highlight the importance of understanding the molecular basis of antibody binding to develop effective therapies. The study suggests that focusing on conserved regions within the RBD-ACE2 interface might be crucial for designing broadly neutralizing antibodies.

This study provides detailed molecular insights into the increased binding affinity and stability of the RBD-ACE2 complex in several SARS-CoV-2 variants of concern. The results demonstrate the significant role of specific mutations in enhancing viral infectivity and potentially evading antibody neutralization. Future research should focus on identifying and targeting conserved regions within the RBD-ACE2 interface to develop broadly effective therapeutics and vaccines against emerging SARS-CoV-2 variants.

The study primarily focuses on a limited set of variants and monoclonal antibodies. While the in vitro and in silico approaches provide valuable insights, the results might not fully capture the complexity of the in vivo interactions. Further studies involving a broader range of variants and antibodies are needed to confirm the generalizability of the findings. The MD simulations used a specific force field; the choice of force field could influence the results. The in vitro conditions might not completely reflect the in vivo cellular environment.