Amphipathic Helices Enable Fatty Acid Uptake and Activation by Human FATP2 at ER Contact Sites

Amphipathic Helices Enable Fatty Acid Uptake and Activation by Human FATP2 at ER Contact Sites | 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 Amphipathic Helices Enable Fatty Acid Uptake and Activation by Human FATP2 at ER Contact Sites Carol Robinson, Haigang Song, Rei Matsuoka, Florencia Klein Rocha, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8895167/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 Fatty acid transport proteins (FATPs) couple fatty acid uptake to metabolic activation; a process frequently dysregulated in metabolic disease and cancer. However, the structural basis of the coupling has remained unclear. Here we combine native mass spectrometry and cryo-EM to capture fatty acyl-adenylate intermediates and determine the first high-resolution structures of a FATP family member, FATP2. The structures reveal two functionally separable modules: an amphipathic helix module enabling membrane-dependent uptake and a catalytic activation module mediating metabolic trapping. Structure-guided mutagenesis demonstrates that the helices are essential for fatty acid uptake, whereas a catalytically inactive splice variant (FATP2b) uncouples membrane-dependent uptake from activation. Molecular dynamics simulations indicate that the two amphipathic helices induce lipid-packing defects and membrane thinning. Consistently, super-resolution live-cell imaging and co-immunoprecipitation show that FATP2 localizes to the endoplasmic reticulum and preferentially associates with ER–organelle contact sites. Together our data establish FATP2 as a prototype for membrane geometry-coupled metabolic enzymes and provide a structural framework for the development of FATP-targeted therapeutics. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Biophysics/Membrane biophysics Biological sciences/Molecular biology/Proteomics/Protein–protein interaction networks Full Text Additional Declarations Yes there is potential Competing Interest. C.V.R. is a cofounder of and scientific advisor at OMass Therapeutics. The other authors declare no competing interests. Supplementary Files cvrFATP2SupplementaryHS260216.pdf Supplementary Material 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-8895167","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":605764378,"identity":"d57da52f-7373-4f9b-9bc6-3ecbb6a1d892","order_by":0,"name":"Carol 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The other authors declare no competing interests.","formattedTitle":"Amphipathic Helices Enable Fatty Acid Uptake and Activation by Human FATP2 at ER Contact Sites","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-8895167/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8895167/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Fatty acid transport proteins (FATPs) couple fatty acid uptake to metabolic activation; a process frequently dysregulated in metabolic disease and cancer. However, the structural basis of the coupling has remained unclear. Here we combine native mass spectrometry and cryo-EM to capture fatty acyl-adenylate intermediates and determine the first high-resolution structures of a FATP family member, FATP2. The structures reveal two functionally separable modules: an amphipathic helix module enabling membrane-dependent uptake and a catalytic activation module mediating metabolic trapping. Structure-guided mutagenesis demonstrates that the helices are essential for fatty acid uptake, whereas a catalytically inactive splice variant (FATP2b) uncouples membrane-dependent uptake from activation. Molecular dynamics simulations indicate that the two amphipathic helices induce lipid-packing defects and membrane thinning. Consistently, super-resolution live-cell imaging and co-immunoprecipitation show that FATP2 localizes to the endoplasmic reticulum and preferentially associates with ER–organelle contact sites. 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