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E channel activity is inhibited by the drug 5\u2010(N,N\u2010hexamethylene) amiloride (HMA). However, the binding site of HMA in E has not been determined. Here we use solid\u2010state NMR to measure distances between HMA and the E transmembrane domain (ETM) in lipid bilayers. <jats:sup>13<\/jats:sup>C, <jats:sup>15<\/jats:sup>N\u2010labeled HMA is combined with fluorinated or <jats:sup>13<\/jats:sup>C\u2010labeled ETM. Conversely, fluorinated HMA is combined with <jats:sup>13<\/jats:sup>C, <jats:sup>15<\/jats:sup>N\u2010labeled ETM. These orthogonal isotopic labeling patterns allow us to conduct dipolar recoupling NMR experiments to determine the HMA binding stoichiometry to ETM as well as HMA\u2010protein distances. We find that HMA binds ETM with a stoichiometry of one drug per pentamer. Unexpectedly, the bound HMA is not centrally located within the channel pore, but lies on the lipid\u2010facing surface in the middle of the TM domain. This result suggests that HMA may inhibit the E channel activity by interfering with the gating function of an aromatic network. These distance data are obtained under much lower drug concentrations than in previous chemical shift perturbation data, which showed the largest perturbation for N\u2010terminal residues. This difference suggests that HMA has higher affinity for the protein\u2013lipid interface than the channel pore. These results give insight into the inhibition mechanism of HMA for SARS\u2010CoV\u20102 E.<\/jats:p>","DOI":"10.1002\/pro.4755","type":"journal-article","created":{"date-parts":[[2023,8,26]],"date-time":"2023-08-26T06:14:20Z","timestamp":1693030460000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Hexamethylene amiloride binds the SARS\u2010CoV\u20102 envelope protein at the protein\u2013lipid interface"],"prefix":"10.1002","volume":"32","author":[{"given":"Noah H.","family":"Somberg","sequence":"first","affiliation":[{"name":"Department of Chemistry Massachusetts Institute of Technology  Cambridge Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jo\u00e3o","family":"Medeiros\u2010Silva","sequence":"additional","affiliation":[{"name":"Department of Chemistry Massachusetts Institute of Technology  Cambridge Massachusetts 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