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N481K RBD mutation emerged early in Qatar (2020) and later re-emerged globally, reaching near-fixation by 2024\u20132025, yet its functional significance remained unexplored. Methods: We analyzed the temporal and geographic circulation of N481K across &gt;\u200917 million SARS-CoV-2 genomes (2020\u20132025). 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\u2009=\u200948) and vaccinated cohorts: mRNA (n\u2009=\u200916), Sinopharm (n\u2009=\u200945), and AstraZeneca (n\u2009=\u200928). Results: N481K was detected in 4.14% (703,777\/17,016,111) of global genomes and reached near-fixation (&gt;\u200990%) 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 \u00c5 RMSD deviation and higher ACE2 docking affinity (\u2013313 vs. \u2212\u2009308 kcal\/mol) compared to the wild-type (WT) RBD. Molecular dynamics confirmed increased stability and lower binding free energy for N481K versus WT (\u201377.07\u2009\u00b1\u20090.90 vs. \u2212\u200943.09\u2009\u00b1\u20090.98 kcal\/mol). Among samples with detectable WT neutralization (n = 89), mean neutralization declined from 69.76% (WT) to 42.47% (N481K) (p\u2009&lt;\u20090.0001), 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.<\/jats:p>","DOI":"10.1007\/s00705-026-06652-y","type":"journal-article","created":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T15:11:15Z","timestamp":1780585875000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Functional and structural characterization of the SARS-CoV-2 spike N481K mutation"],"prefix":"10.1007","volume":"171","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7621-0130","authenticated-orcid":false,"given":"Maria K.","family":"Smatti","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hebah","family":"Al-Khatib","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhammad","family":"Suleman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muna","family":"Nizar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hala Abo","family":"Khalid","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruaa","family":"Ismail","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gheyath K.","family":"Nasrallah","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hadi M.","family":"Yassine","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,6,4]]},"reference":[{"issue":"7898","key":"6652_CR1","doi-asserted-by":"publisher","first-page":"657","DOI":"10.1038\/s41586-021-04385-3","volume":"602","author":"Y Cao","year":"2022","unstructured":"Cao Y, Wang J, Jian F, Xiao T, Song W, Yisimayi A et al (2022) Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. 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