Structural and biochemical basis of ROC-dependent activation of LRRK2

Structural and biochemical basis of ROC-dependent activation of LRRK2 | 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 and biochemical basis of ROC-dependent activation of LRRK2 Quyen Hoang, Yangshin Park, Chunxiang Wu, Kayla Tennessen, Li Wan, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8735353/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Apr, 2026 Read the published version in PNAS Nexus → Version 1 posted You are reading this latest preprint version Abstract Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease, yet the molecular mechanism governing LRRK2 activation remains incompletely understood. LRRK2 is a large multidomain enzyme whose kinase activity is regulated by intramolecular interactions and by its Ras of complex proteins (ROC) GTPase domain. Here, we combine cryo–electron microscopy, X-ray crystallography, and structure-guided biochemical perturbations to define how ROC conformational switching regulates LRRK2 activation. Cryo-EM reconstructions reveal that monomeric full-length LRRK2 samples three distinct conformational states—autoinhibited, intermediate, and activated—indicating that large-scale activation-associated rearrangements can occur through an intrinsic intramolecular pathway, independently of Rab29 binding, higher-order oligomerization, or membrane association. A 1.6 Å crystal structure of an extended ROC construct reveals intrinsic conformational plasticity within the GTPase switch regions that likely underlies these transitions. Structure-guided disulfide engineering identifies a functional coupling between residue R1441 and Switch II that directly modulates GTPase activity in both isolated ROC and full-length LRRK2. Disruption of this coupling phenocopies the disease-associated R1441H mutation. Together, these findings establish ROC as a dynamic conformational engine that drives a multistep intramolecular activation mechanism in LRRK2, providing mechanistic insight into how pathogenic mutations promote aberrant kinase activation. Biological sciences/Structural biology/X-ray crystallography Biological sciences/Biochemistry/Enzyme mechanisms Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy LRRK2 Parkinson’s disease GTPase Kinase X-ray crystallography Cryo-EM Full Text Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Published Journal Publication published 08 Apr, 2026 Read the published version in PNAS Nexus → 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-8735353","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":583136726,"identity":"d6d8a590-929f-4a85-9bc3-df0257418530","order_by":0,"name":"Quyen 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Cryo-EM","lastPublishedDoi":"10.21203/rs.3.rs-8735353/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8735353/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease, yet the molecular mechanism governing LRRK2 activation remains incompletely understood. LRRK2 is a large multidomain enzyme whose kinase activity is regulated by intramolecular interactions and by its Ras of complex proteins (ROC) GTPase domain. Here, we combine cryo–electron microscopy, X-ray crystallography, and structure-guided biochemical perturbations to define how ROC conformational switching regulates LRRK2 activation. Cryo-EM reconstructions reveal that monomeric full-length LRRK2 samples three distinct conformational states—autoinhibited, intermediate, and activated—indicating that large-scale activation-associated rearrangements can occur through an intrinsic intramolecular pathway, independently of Rab29 binding, higher-order oligomerization, or membrane association. A 1.6 Å crystal structure of an extended ROC construct reveals intrinsic conformational plasticity within the GTPase switch regions that likely underlies these transitions. Structure-guided disulfide engineering identifies a functional coupling between residue R1441 and Switch II that directly modulates GTPase activity in both isolated ROC and full-length LRRK2. Disruption of this coupling phenocopies the disease-associated R1441H mutation. Together, these findings establish ROC as a dynamic conformational engine that drives a multistep intramolecular activation mechanism in LRRK2, providing mechanistic insight into how pathogenic mutations promote aberrant kinase activation.","manuscriptTitle":"Structural and biochemical basis of ROC-dependent activation of LRRK2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 06:00:35","doi":"10.21203/rs.3.rs-8735353/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d562277e-a419-4f70-9b6f-3e81a78d2dd6","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":62044482,"name":"Biological sciences/Structural biology/X-ray crystallography"},{"id":62044483,"name":"Biological sciences/Biochemistry/Enzyme mechanisms"},{"id":62044484,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"}],"tags":[],"updatedAt":"2026-04-14T14:58:24+00:00","versionOfRecord":{"articleIdentity":"rs-8735353","link":"https://doi.org/10.1093/pnasnexus/pgag112","journal":{"identity":"pnas-nexus","isVorOnly":true,"title":"PNAS Nexus"},"publishedOn":"2026-04-09 00:00:00","publishedOnDateReadable":"April 9th, 2026"},"versionCreatedAt":"2026-02-02 06:00:35","video":"","vorDoi":"10.1093/pnasnexus/pgag112","vorDoiUrl":"https://doi.org/10.1093/pnasnexus/pgag112","workflowStages":[]},"version":"v1","identity":"rs-8735353","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8735353","identity":"rs-8735353","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}