Structural basis of metalloid transport by the arsenite efflux pump ArsB

Structural basis of metalloid transport by the arsenite efflux pump ArsB | 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 Structural basis of metalloid transport by the arsenite efflux pump ArsB William Clemons, Shivansh Mahajan, Kemal Demirer, Douglas Rees This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8921025/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 Bacteria resist toxic arsenite (AsIII) in their environments by actively pumping the metalloid out of the cell via efflux pumps such as ArsB. However, the mechanism of extrusion remains poorly understood, which hinders the development of engineered bioremediation strategies. We report high-resolution cryo-EM structures of ArsB from the arsenic-tolerant bacterium Leptospirillum ferriphilum. ArsB adopts an inverted two-fold repeat architecture resembling that of other ion transporter (IT) superfamily proteins. Structures determined in the presence of As III and antimonite (Sb III ) reveal that the metalloid substrates interact with polar residues at the core of the transmembrane domain primarily via hydrogen bonding. Mutagenesis and in vivo functional assays support these interactions. Our ArsB structures represent an ‘inward-facing’ conformation, where the metalloid-binding site is exposed to the cytoplasm, suitable for metalloid capture. Furthermore, we demonstrate that As III resistance conferred by ArsB varies with external pH, supporting that ArsB is a proton (H+)-coupled secondary transporter. Mutagenesis, in vivo functional assays, and pKa estimation imply that conserved aspartate residues near the metalloid-binding site likely mediate the H+-coupling mechanism. Our findings provide structural insights into metalloid recognition and H+/metalloid antiport in ArsB, laying a foundation for further elucidation of the molecular basis of toxic metalloid detoxification in bacteria. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Microbiology/Bacteria/Bacterial structural biology Biological sciences/Biochemistry/Proteins/Membrane proteins Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryinformationsm.pdf Supplementary Tables and Figures 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-8921025","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":596459730,"identity":"ad0bf924-5212-4126-a5d3-3cd18de09626","order_by":0,"name":"William Clemons","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYDACdgglx8DA3ACkDwBxAgEtzBDKmIGBsbGBJC2JDURr4W9mPrrh44669P72g+0PPtTcYeBnzzHAq0XiMFvazZlnDufOOJPY2Djj2DMGyZ43+LUYMPOY3eZtO5C7gSGxsZm34TCDwQ0Cthgw838DaqlLN+B/2Nj8F6jFnrAWHjagFuYEAwmgLYwgWyQI+8Xs5sy2w4YzbjxsnNlz7DCPxJlnBXi18Lc3P7vxsa1Onr8/+cCHHzWH5fjbkzfg1YIBeEhTPgpGwSgYBaMAKwAArQlMjR9WGkcAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0021-889X","institution":"California Institute of Technology","correspondingAuthor":true,"prefix":"","firstName":"William","middleName":"","lastName":"Clemons","suffix":""},{"id":596459731,"identity":"9951ea93-761c-48d0-ac8f-869cc245a9b2","order_by":1,"name":"Shivansh Mahajan","email":"","orcid":"https://orcid.org/0000-0002-3041-7988","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shivansh","middleName":"","lastName":"Mahajan","suffix":""},{"id":596459732,"identity":"5973ead3-0573-4149-967f-5e02a76eae9f","order_by":2,"name":"Kemal Demirer","email":"","orcid":"","institution":"California Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Kemal","middleName":"","lastName":"Demirer","suffix":""},{"id":596459733,"identity":"19871597-3685-42c4-bbaf-3c38c74ce87c","order_by":3,"name":"Douglas Rees","email":"","orcid":"https://orcid.org/0000-0003-4073-1185","institution":"California Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Douglas","middleName":"","lastName":"Rees","suffix":""}],"badges":[],"createdAt":"2026-02-19 22:05:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8921025/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8921025/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103388571,"identity":"e43987cb-da26-445d-b830-cac842f1836c","added_by":"auto","created_at":"2026-02-25 07:13:35","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3965874,"visible":true,"origin":"","legend":"Article File","description":"","filename":"ArsBvfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8921025/v1_covered_b30fd545-96af-4b6f-98ea-2bfd021ca80b.pdf"},{"id":103388446,"identity":"aad3d344-87e5-43fa-a26d-3aec9fbad7a3","added_by":"auto","created_at":"2026-02-25 07:13:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6360954,"visible":true,"origin":"","legend":"Supplementary Tables and Figures","description":"","filename":"Supplementaryinformationsm.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8921025/v1/ac8add56212e0e8b195fc81b.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Structural basis of metalloid transport by the arsenite efflux pump ArsB","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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-8921025/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8921025/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Bacteria resist toxic arsenite (AsIII) in their environments by actively pumping the metalloid out of the cell via efflux pumps such as ArsB. However, the mechanism of extrusion remains poorly understood, which hinders the development of engineered bioremediation strategies. We report high-resolution cryo-EM structures of ArsB from the arsenic-tolerant bacterium Leptospirillum ferriphilum. ArsB adopts an inverted two-fold repeat architecture resembling that of other ion transporter (IT) superfamily proteins. Structures determined in the presence of As\u003csup\u003eIII\u003c/sup\u003e and antimonite (Sb\u003csup\u003eIII\u003c/sup\u003e) reveal that the metalloid substrates interact with polar residues at the core of the transmembrane domain primarily via hydrogen bonding. Mutagenesis and in vivo functional assays support these interactions. Our ArsB structures represent an ‘inward-facing’ conformation, where the metalloid-binding site is exposed to the cytoplasm, suitable for metalloid capture. Furthermore, we demonstrate that As\u003csup\u003eIII\u003c/sup\u003e resistance conferred by ArsB varies with external pH, supporting that ArsB is a proton (H+)-coupled secondary transporter. Mutagenesis, in vivo functional assays, and pKa estimation imply that conserved aspartate residues near the metalloid-binding site likely mediate the H+-coupling mechanism. Our findings provide structural insights into metalloid recognition and H+/metalloid antiport in ArsB, laying a foundation for further elucidation of the molecular basis of toxic metalloid detoxification in bacteria.","manuscriptTitle":"Structural basis of metalloid transport by the arsenite efflux pump ArsB","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-25 07:10:57","doi":"10.21203/rs.3.rs-8921025/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":"cf2d041b-bc42-4453-b94b-b8154c908d71","owner":[],"postedDate":"February 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63468046,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":63468047,"name":"Biological sciences/Microbiology/Bacteria/Bacterial structural biology"},{"id":63468048,"name":"Biological sciences/Biochemistry/Proteins/Membrane proteins"}],"tags":[],"updatedAt":"2026-05-07T17:00:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-25 07:10:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8921025","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8921025","identity":"rs-8921025","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}