Antibiotic-Specific Conformational Landscapes of a Multidrug Transporter

Antibiotic-Specific Conformational Landscapes of a Multidrug Transporter | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Antibiotic-Specific Conformational Landscapes of a Multidrug Transporter Chloe Martens, Maklad Hassan, Tom Kache, Aurélie Roth, Maria Mamkaeva, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9357337/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Multidrug transporters are membrane proteins that can transport an ensemble of structurally dissimilar compounds and contribute to bacterial multidrug resistance (MDR) by exporting different antibiotics from the cell. However, whether they transport different substrates through a common mechanism or via distinct substrate-dependent mechanisms remains unclear. In this work, we used single-molecule Förster resonance energy transfer (smFRET) to measure time-resolved conformational dynamics of LmrP, a multidrug transporter of the Major Facilitator Superfamily (MFS). We present high-resolution conformational landscapes of LmrP in the presence of different antibiotics. Through multi-parameter Hidden Markov Modeling (mpH2MM), we uncovered transient states and quantified their sub-millisecond interconversion kinetics. We observed antibiotic-dependent heterogeneity in the conformational landscape, both in accessible states and in interconversion rates. Notably, poorly or non-transported antibiotics slow down transition kinetics, pointing to rapid state interconversion as a driver of efficient transport. This suggests that MFS MDR transporters bind and export structurally dissimilar antibiotics by relying on an array of underlying conformational states with ligand-dictated interconversion rates. This work provides novel insights into the mechanism of MDR transporters and advocates for combined structure/dynamics-based drug design when targeting their function. Biological sciences/Structural biology Biological sciences/Biophysics/Single-molecule biophysics Biological sciences/Biophysics/Permeation and transport Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementarymaterialLmrPver8April2026.pdf Supplementary Information Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9357337","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":635728663,"identity":"86c3fd78-15a0-4801-9601-ab06c049e56e","order_by":0,"name":"Chloe Martens","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYDACCeYGJF4Fg4wBAw8hLYxQLWwg4gwDD4laGNuI0MI/u7HxwQeGbfLm85uPPfw57zCPOQPvwQd4LblzsNlwBsNtwznH2NKNebcd5rFs4Es2wKfFQCKxTZqH4TbjDDYeM2lGoBaDAzxmEgS1/GG4bT+Djf+b5M85YC3mPwhqYWC4nQi0hU2CtwFiCz4dDBI3EpsNewxuJ89gSzOT5jmWzmPZzJeM12H8M5IPPvhRcdt2BvPhZ5I/aqzlzNl7D37Aaw3EecgcZsLqR8EoGAWjYBQQAACfF0KS0Dm90QAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1617-4238","institution":"Université Libre de Bruxelles","correspondingAuthor":true,"prefix":"","firstName":"Chloe","middleName":"","lastName":"Martens","suffix":""},{"id":635728664,"identity":"4fe1b93a-259b-4a41-a253-baaf31d67229","order_by":1,"name":"Maklad Hassan","email":"","orcid":"","institution":"Université Libre de Bruxelles","correspondingAuthor":false,"prefix":"","firstName":"Maklad","middleName":"","lastName":"Hassan","suffix":""},{"id":635728665,"identity":"9e04e049-12b6-4516-b375-a40e73191e95","order_by":2,"name":"Tom Kache","email":"","orcid":"","institution":"Hasselt University","correspondingAuthor":false,"prefix":"","firstName":"Tom","middleName":"","lastName":"Kache","suffix":""},{"id":635728666,"identity":"1288caa4-4c8f-4cee-8d64-7aa91718bb55","order_by":3,"name":"Aurélie Roth","email":"","orcid":"","institution":"Université Libre de Bruxelles","correspondingAuthor":false,"prefix":"","firstName":"Aurélie","middleName":"","lastName":"Roth","suffix":""},{"id":635728667,"identity":"1ac3f2ca-5854-4b79-a614-e8c29e97c25c","order_by":4,"name":"Maria Mamkaeva","email":"","orcid":"","institution":"Université Libre de Bruxelles","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Mamkaeva","suffix":""},{"id":635728668,"identity":"4ee044f7-23ce-4075-bf56-93e641039b7c","order_by":5,"name":"Cédric Govaerts","email":"","orcid":"https://orcid.org/0000-0003-3218-3262","institution":"ULB","correspondingAuthor":false,"prefix":"","firstName":"Cédric","middleName":"","lastName":"Govaerts","suffix":""},{"id":635728669,"identity":"97abad80-04bb-4ac7-b338-5c84a5439b4d","order_by":6,"name":"Jelle Hendrix","email":"","orcid":"https://orcid.org/0000-0001-5731-1297","institution":"Hasselt University","correspondingAuthor":false,"prefix":"","firstName":"Jelle","middleName":"","lastName":"Hendrix","suffix":""}],"badges":[],"createdAt":"2026-04-08 13:02:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9357337/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9357337/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109205139,"identity":"435e10fa-6ba4-4a0d-9852-ccc14d433fcd","added_by":"auto","created_at":"2026-05-13 15:03:29","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12272738,"visible":true,"origin":"","legend":"Article File","description":"","filename":"LmrPManuscriptVer8April26FinalCMdriveCopy.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9357337/v1_covered_bdbef41c-6b04-4691-8f99-d4f13ef0ed01.pdf"},{"id":109119436,"identity":"6ec9436e-ab6d-451c-a924-3a63e06fc76d","added_by":"auto","created_at":"2026-05-12 16:57:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1718935,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementarymaterialLmrPver8April2026.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9357337/v1/1e17fc35ef1e0a39e77c72b8.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Antibiotic-Specific Conformational Landscapes of a Multidrug Transporter","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9357337/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9357337/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Multidrug transporters are membrane proteins that can transport an ensemble of structurally dissimilar compounds and contribute to bacterial multidrug resistance (MDR) by exporting different antibiotics from the cell. However, whether they transport different substrates through a common mechanism or via distinct substrate-dependent mechanisms remains unclear. In this work, we used single-molecule Förster resonance energy transfer (smFRET) to measure time-resolved conformational dynamics of LmrP, a multidrug transporter of the Major Facilitator Superfamily (MFS). We present high-resolution conformational landscapes of LmrP in the presence of different antibiotics. Through multi-parameter Hidden Markov Modeling (mpH2MM), we uncovered transient states and quantified their sub-millisecond interconversion kinetics. We observed antibiotic-dependent heterogeneity in the conformational landscape, both in accessible states and in interconversion rates. Notably, poorly or non-transported antibiotics slow down transition kinetics, pointing to rapid state interconversion as a driver of efficient transport. This suggests that MFS MDR transporters bind and export structurally dissimilar antibiotics by relying on an array of underlying conformational states with ligand-dictated interconversion rates. This work provides novel insights into the mechanism of MDR transporters and advocates for combined structure/dynamics-based drug design when targeting their function.","manuscriptTitle":"Antibiotic-Specific Conformational Landscapes of a Multidrug Transporter","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-12 16:57:32","doi":"10.21203/rs.3.rs-9357337/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bc17f9a5-cfc1-40b6-9c37-beaa585ea6e2","owner":[],"postedDate":"May 12th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-07T07:50:09+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-07T06:18:41+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"5","date":"2026-05-06T19:17:24+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67662533,"name":"Biological sciences/Structural biology"},{"id":67662534,"name":"Biological sciences/Biophysics/Single-molecule biophysics"},{"id":67662535,"name":"Biological sciences/Biophysics/Permeation and transport"}],"tags":[],"updatedAt":"2026-05-12T16:57:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-12 16:57:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9357337","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9357337","identity":"rs-9357337","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}