From Alpha to Omicron: Structural Insights into SARS-CoV-2 RBD Evolution and ACE2 Binding

Abstract Background Major variants of SARS-CoV-2 have significantly altered transmissibility and infection characteristics, posing substantial challenges to public health. In particular, structural changes in the receptor-binding domain (RBD) of the spike protein influence its binding affinity to the human ACE2 receptor. This study aimed to quantitatively assess differences in RBD features and ACE2-binding properties across SARS-CoV-2 variants to better understand their public health implications. Methods Spike protein sequences from five major SARS-CoV-2 variants-Alpha, Beta, Gamma, Delta, and Omicron-were analysed. Full-length amino acid sequences and RBD regions were extracted and examined through multiple sequence alignment and conservation scoring. Key mutations were identified, and protein-protein docking was carried out to evaluate binding interactions between variant RBDs and human ACE2. Docking scores and confidence scores were compared. Additionally, molecular dynamics (MD) simulations were performed to assess the structural stability of the RBD-ACE2 complexes, with root mean square fluctuation (RMSF) used to quantify flexibility at the binding interface. Results Omicron exhibited the highest number of amino acid substitutions within the RBD and the lowest sequence conservation. Mutations such as N501Y, L452R, and E484K were identified, potentially contributing to binding affinity and immune evasion. Docking analysis showed that Omicron had the strongest predicted binding to ACE2, followed by Alpha and Delta. MD simulations revealed that Gamma exhibited the highest structural flexibility, whereas Alpha and Omicron maintained relatively stable interactions. Conclusions This study provides an integrated comparison of RBD structure and ACE2-binding characteristics among SARS-CoV-2 variants. The structural properties of the variants contribute to differences in transmissibility and may inform variant monitoring and public health response strategies. Key messages • Structural analysis of SARS-CoV-2 variants supports surveillance and public health planning. • Omicron harbors the highest number of RBD substitutions and exhibits the strongest ACE2 binding.