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While mutations in the receptor-binding domain (RBD) enhance binding to the ACE2 receptor and evade neutralizing antibodies, the function of mutations in the N-terminal domain (NTD) remains poorly understood. Using two independent methods, surface plasmon resonance (SPR) and cryo-EM, we show that NTD mutations increase the population of spike protein with the RBD in the \u201cup\u201d conformational state. SPR association and dissociation kinetics of spike binding to ACE2 and antibodies, analyzed using a coupled equilibrium model, determined the relative populations and indicated that the RBD-up-to-down transition is rate-limiting relative to the RBD-down-to-up transition. Advanced model fitting of cryo-EM Coulomb potential maps confirmed the populations. The combined effect of NTD and RBD mutations exceeds the sum of their individual effects, indicating long-range allosteric communication and energetic coupling within the spike protein.<\/jats:p>","DOI":"10.64898\/2026.04.20.719543","type":"posted-content","created":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T03:45:22Z","timestamp":1776915922000},"source":"Crossref","is-referenced-by-count":0,"title":["Long-range allosteric communication, double mutant cycles, and energetic coupling in SARS-CoV-2 spike protein"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7655-3590","authenticated-orcid":false,"given":"Alexandra","family":"Lucas","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2823-2984","authenticated-orcid":false,"given":"Vamseedhar","family":"Rayaprolu","sequence":"additional","affiliation":[{"name":"Pacific Northwest Cryo-EM Center, Oregon Health & Science University, Portland, OR, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8308-5318","authenticated-orcid":false,"given":"Krishna M.G.","family":"Mallela","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026042512351794000_2026.04.20.719543v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-018-0118-9"},{"key":"2026042512351794000_2026.04.20.719543v1.2","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1146\/annurev-virology-110615-042301","article-title":"Structure, Function, and Evolution of Coronavirus Spike Proteins","volume":"3","year":"2016","journal-title":"Annu Rev Virol"},{"key":"2026042512351794000_2026.04.20.719543v1.3","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.med.56.091103.134135"},{"key":"2026042512351794000_2026.04.20.719543v1.4","first-page":"34","article-title":"Why was the sars-cov epidemic (2002-2003) so rapidly contained while sars-cov-2 easily spread since december 2019 and gave rise to the actual pandemic?","volume":"3","year":"2021","journal-title":"Electron J Biomed"},{"key":"2026042512351794000_2026.04.20.719543v1.5","unstructured":"21 million deaths - Over 7m Covid-19 fatalities recorded, but actual number may be 3 times higher: WHO. 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