Distinct transport cycle and lipid regulation of a Mg2+-transporting P-type ATPase, MgtA

Distinct transport cycle and lipid regulation of a Mg2+-transporting P-type ATPase, MgtA | 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 Distinct transport cycle and lipid regulation of a Mg 2+ -transporting P-type ATPase, MgtA Howard Young, Muhammad Bashir Khan, Joseph Primeau, Paramita Chaudhuri-Basu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9173029/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 P-type ATPases represent an evolutionarily conserved superfamily of ion, lipid, and peptide pumps found across all domains of life. Among the substrates transported by P-type ATPases, Mg2+ is of critical importance in bacterial, fungal, and plant cellular homeostasis. A bacterial P-type ATPase found in Gram-negative bacteria, Mg2+ transporter A (MgtA), facilitates the transport of Mg2+ from the periplasm to the cytoplasm under conditions of Mg2+ starvation. MgtA is a cardiolipin-sensitive integral membrane ion-transporter that scavenges Mg2+ during bacterial infection and pathogenesis. Here, we determined cryo-EM structures of MgtA capturing three distinct states along the Mg2+ transport cycle, including a phosphorylated E2-P intermediate (2.6 Å resolution), an E1-like conformation stabilized by the peptide regulator MgtR (2.7 Å resolution), and an E1-like ATP-bound state (2.8 Å resolution). These three conformations reveal the binding of Mg2+ in the transmembrane domain coordinated in a novel site involving Ser702 and Asn706 on M5, Ser773 and Asp777 on M7, and Ser821 and Thr824 on M8. In the E2-P conformation, the phosphate analog BeF3 is bound in close proximity to the catalytic aspartate, Asp361, suggesting that it represents a covalent aspartylphosphate intermediate. In the presence of AMPPCP, Mg2+ remains bound in the transmembrane domain and the ATP analog is bound in a catalytically competent conformation. Overall, the structures reveal distinct steps in the transport cycle of MgtA compared to other P-type ATPases, as well as lipid binding sites that fill gaps in our understanding of transport regulation. Biological sciences/Structural biology Biological sciences/Biophysics/Molecular biophysics Full Text Additional Declarations There is NO Competing Interest. 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. 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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-9173029","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":616659893,"identity":"b7ed2c76-6d88-4647-8fd8-32130a1344e2","order_by":0,"name":"Howard Young","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBACAwhlw8DAzkOaljQGBmaQlgTitRwmQYu5RPIx6YJf5xP7m3kPPq78YcfA334AvxbLGWlp0jP7bifOOMyXbHgmIZlB4gwBqwxu5JhJ8/bcTmw4zGMm2ZDADHQqcVrOJc4/zGP+syGhnsGA/wERWnh+HEjcALSFsSHhMIOBBCFbzjxLtuZtSDbeCPSLZEPacR6JG4RsOZ588DbPHzvZecd7D35ssKmW4+8nYAuDAFABYxuCT0Qa4D8AJP4QVjcKRsEoGAUjGAAAWWdDTjep4WQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5990-8422","institution":"University of Alberta","correspondingAuthor":true,"prefix":"","firstName":"Howard","middleName":"","lastName":"Young","suffix":""},{"id":616659894,"identity":"8fe3937f-1125-4259-805b-d883d5c3bd93","order_by":1,"name":"Muhammad Bashir Khan","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Bashir","lastName":"Khan","suffix":""},{"id":616659895,"identity":"e870d33f-1aec-400a-b343-2247f7311505","order_by":2,"name":"Joseph Primeau","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"","lastName":"Primeau","suffix":""},{"id":616659896,"identity":"796514b4-6e2d-4835-a92f-8193e9bcbd46","order_by":3,"name":"Paramita Chaudhuri-Basu","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Paramita","middleName":"","lastName":"Chaudhuri-Basu","suffix":""},{"id":616659897,"identity":"4d46fd16-4bde-4bb7-a0dd-32e61726646f","order_by":4,"name":"Lucie Bergdoll","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Lucie","middleName":"","lastName":"Bergdoll","suffix":""},{"id":616659898,"identity":"e4b1a2e4-15b8-4c51-98c6-2929c7070a10","order_by":5,"name":"Ludovic Renault","email":"","orcid":"https://orcid.org/0000-0001-6464-8808","institution":"Leiden University","correspondingAuthor":false,"prefix":"","firstName":"Ludovic","middleName":"","lastName":"Renault","suffix":""},{"id":616659899,"identity":"c18a0f26-3b21-451a-a96f-0953d8da3e78","order_by":6,"name":"J. 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Among the substrates transported by P-type ATPases, Mg2+ is of critical importance in bacterial, fungal, and plant cellular homeostasis. A bacterial P-type ATPase found in Gram-negative bacteria, Mg2+ transporter A (MgtA), facilitates the transport of Mg2+ from the periplasm to the cytoplasm under conditions of Mg2+ starvation. MgtA is a cardiolipin-sensitive integral membrane ion-transporter that scavenges Mg2+ during bacterial infection and pathogenesis. Here, we determined cryo-EM structures of MgtA capturing three distinct states along the Mg2+ transport cycle, including a phosphorylated E2-P intermediate (2.6 Å resolution), an E1-like conformation stabilized by the peptide regulator MgtR (2.7 Å resolution), and an E1-like ATP-bound state (2.8 Å resolution). These three conformations reveal the binding of Mg2+ in the transmembrane domain coordinated in a novel site involving Ser702 and Asn706 on M5, Ser773 and Asp777 on M7, and Ser821 and Thr824 on M8. In the E2-P conformation, the phosphate analog BeF3 is bound in close proximity to the catalytic aspartate, Asp361, suggesting that it represents a covalent aspartylphosphate intermediate. In the presence of AMPPCP, Mg2+ remains bound in the transmembrane domain and the ATP analog is bound in a catalytically competent conformation. Overall, the structures reveal distinct steps in the transport cycle of MgtA compared to other P-type ATPases, as well as lipid binding sites that fill gaps in our understanding of transport regulation.","manuscriptTitle":"Distinct transport cycle and lipid regulation of a Mg2+-transporting P-type ATPase, MgtA","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 11:46:56","doi":"10.21203/rs.3.rs-9173029/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":"0f4a9515-1f52-452f-8248-520620a55175","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67480299,"name":"Biological sciences/Structural biology"},{"id":67480300,"name":"Biological sciences/Biophysics/Molecular biophysics"}],"tags":[],"updatedAt":"2026-05-04T11:46:56+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 11:46:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9173029","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9173029","identity":"rs-9173029","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}