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Multidrug efflux pumps of the resistance nodulation and cell division (RND) superfamily function as part of cell envelope spanning systems and provide resistance to diverse antibiotics. Here, we identify two phylogenetic clusters of RND proteins with conserved binding pocket residues and show that the transfer of a single conserved residue between both clusters affects the resistance phenotype not only due to changes in the physicochemical properties of the binding pocket, but also due to an altered equilibrium between the conformational states of the transport cycle. We demonstrate, using single-particle cryo-electron microscopy, that AcrB and OqxB, which represent both clusters, adopt fundamentally different apo states, implying distinct mechanisms for initial substrate binding. The observed conformational plasticity appears phylogenetically conserved and likely plays a role in the diversification of the resistance phenotype among homologous RND pumps.<\/jats:p>","DOI":"10.1038\/s41467-025-66751-3","type":"journal-article","created":{"date-parts":[[2025,11,26]],"date-time":"2025-11-26T15:37:42Z","timestamp":1764171462000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Conformational plasticity across phylogenetic clusters of RND multidrug efflux pumps and its impact on substrate specificity"],"prefix":"10.1038","volume":"16","author":[{"given":"Mariya","family":"Lazarova","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Eicher","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clara","family":"B\u00f6rnsen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hui","family":"Zeng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6337-1026","authenticated-orcid":false,"given":"Mohd","family":"Athar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7633-9308","authenticated-orcid":false,"given":"Ui","family":"Okada","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4278-0039","authenticated-orcid":false,"given":"Eiki","family":"Yamashita","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Inga M.","family":"Spannaus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Max","family":"Borgosch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hi-jea","family":"Cha","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4013-8867","authenticated-orcid":false,"given":"Attilio V.","family":"Vargiu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5553-7663","authenticated-orcid":false,"given":"Satoshi","family":"Murakami","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5360-0905","authenticated-orcid":false,"given":"Kay","family":"Diederichs","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Achilleas S.","family":"Frangakis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9035-3827","authenticated-orcid":false,"given":"Klaas M.","family":"Pos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,11,26]]},"reference":[{"key":"66751_CR1","doi-asserted-by":"publisher","first-page":"280","DOI":"10.1038\/s41579-022-00820-y","volume":"21","author":"EM Darby","year":"2023","unstructured":"Darby, E. 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