Functional and structural characterization of the SARS-CoV-2 spike N481K mutation

Background: Selective pressure drives SARS-CoV-2 evolution, with spike RBD substitutions shaping ACE2 binding and antibody escape. N481K RBD mutation emerged early in Qatar (2020) and later re-emerged globally, reaching near-fixation by 2024–2025, yet its functional significance remained unexplored. Methods: We analyzed the temporal and geographic circulation of N481K across > 17 million SARS-CoV-2 genomes (2020–2025). Structural effects were assessed using modeling, docking, and molecular dynamics. Functional impact was evaluated using VSV pseudovirus neutralization tests with sera from infected individuals (n = 48) and vaccinated cohorts: mRNA (n = 16), Sinopharm (n = 45), and AstraZeneca (n = 28). Results: N481K was detected in 4.14% (703,777/17,016,111) of global genomes and reached near-fixation (> 90%) in dominant Omicron lineages by 2024, with prevalence rising from 71.5% in early 2024 to 94.4% by week 36 of 2025. Structural analyses showed a 0.706 Å RMSD deviation and higher ACE2 docking affinity (–313 vs. − 308 kcal/mol) compared to the wild-type (WT) RBD. Molecular dynamics confirmed increased stability and lower binding free energy for N481K versus WT (–77.07 ± 0.90 vs. − 43.09 ± 0.98 kcal/mol). Among samples with detectable WT neutralization (n = 89), mean neutralization declined from 69.76% (WT) to 42.47% (N481K) (p < 0.0001), with the largest reductions in AstraZeneca, Sinopharm, and infected cohorts. mRNA recipients retained the highest neutralization [77.5% (WT) vs. 56.0% (N481K)]. Conclusion: N481K enhances ACE2 binding while partially evading antibody neutralization, suggesting viral fitness and persistence.