Mechanism of activation of β-1,3-glucan synthase by Rho1

Mechanism of activation of β-1,3-glucan synthase by Rho1 | 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 Mechanism of activation of β-1,3-glucan synthase by Rho1 Xiang Wang, Jialu Li, Huayi Liu, Jian Li, Juxiu Liu, Xinli Dai, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3845625/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Feb, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The β-1,3 glucan synthase (GS) is essential for the biosynthesis of β-1,3 glucan, a well-conserved structural component of fungal cell wall. The GS holoenzyme is a multi-enzyme complex consisting of the glycosyltransferase FKS and the essential regulatory factor Rho1, a small GTPase. However, the precise mechanism by which Rho1 activates FKS1 activity in a GTP-dependent manner remains elusive. Here, we present two cryo-electron microscopy (cryo-EM) structures of FKS1 alone (resting state) and FKS1-Rho1 complex (activating state), respectively. Structural analysis reveals that FKS1 adopts a cellulase-like conformation, wherein two segments of the cytoplasmic domain tightly bound together to form a functional structural unit. Remarkably, we unveil that the interaction between Rho1 and FKS1 is enhanced in the presence of a nonhydrolyzable guanosine triphosphate analog (GTP-γ-S). Rho1 is positioned within a pocket between the cytoplasmic domain of FKS1 and the transmembrane helix spanning TM7-15, engaging with the highly conserved glycosyltransferase domain of FKS1 (GT domain). Comparative analysis between the unbound (resting state) and Rho1-bound structures of FKS1 reveals the extensive conformational changes within FKS1, specifically in the GT domain and TM7-15. These alterations suggest that Rho1's GTP/GDP cycling acts as a molecular pump, inducing a dynamic transition between the resting and activating states of FKS1. Notably, the activation of Rho1 triggering FKS1 conformation changes, an evolutionary conserved "finger helix" within the FKS1-Rho1 complex adopts an up-and-down movement, ultimately pushing the growing glucan chain into FKS1’s transmembrane channel, thereby facilitating β-1,3 glucan elongation. Collectively, our results provide a vivid ratchet and pawl model to describe the mechanism of fungal β-1,3-glucan biosynthesis. Biological sciences/Biophysics/Membrane structure and assembly Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Full Text Additional Declarations There is NO Competing Interest. Supplementary Files moviwS1FINAL.mov Mechanism of activation of β-1,3-glucan synthase by Rho1 Cite Share Download PDF Status: Published Journal Publication published 28 Feb, 2025 Read the published version in Nature Communications → 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-3845625","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":272037928,"identity":"1c8226df-486d-4422-91f0-edf88ad41cac","order_by":0,"name":"Xiang 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Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3845625/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3845625/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The β-1,3 glucan synthase (GS) is essential for the biosynthesis of β-1,3 glucan, a well-conserved structural component of fungal cell wall. The GS holoenzyme is a multi-enzyme complex consisting of the glycosyltransferase FKS and the essential regulatory factor Rho1, a small GTPase. However, the precise mechanism by which Rho1 activates FKS1 activity in a GTP-dependent manner remains elusive. Here, we present two cryo-electron microscopy (cryo-EM) structures of FKS1 alone (resting state) and FKS1-Rho1 complex (activating state), respectively. Structural analysis reveals that FKS1 adopts a cellulase-like conformation, wherein two segments of the cytoplasmic domain tightly bound together to form a functional structural unit. Remarkably, we unveil that the interaction between Rho1 and FKS1 is enhanced in the presence of a nonhydrolyzable guanosine triphosphate analog (GTP-γ-S). Rho1 is positioned within a pocket between the cytoplasmic domain of FKS1 and the transmembrane helix spanning TM7-15, engaging with the highly conserved glycosyltransferase domain of FKS1 (GT domain). Comparative analysis between the unbound (resting state) and Rho1-bound structures of FKS1 reveals the extensive conformational changes within FKS1, specifically in the GT domain and TM7-15. These alterations suggest that Rho1's GTP/GDP cycling acts as a molecular pump, inducing a dynamic transition between the resting and activating states of FKS1. Notably, the activation of Rho1 triggering FKS1 conformation changes, an evolutionary conserved \"finger helix\" within the FKS1-Rho1 complex adopts an up-and-down movement, ultimately pushing the growing glucan chain into FKS1’s transmembrane channel, thereby facilitating β-1,3 glucan elongation. Collectively, our results provide a vivid ratchet and pawl model to describe the mechanism of fungal β-1,3-glucan biosynthesis.","manuscriptTitle":"Mechanism of activation of β-1,3-glucan synthase by Rho1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-12 02:13:59","doi":"10.21203/rs.3.rs-3845625/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":"e006bebb-5536-4dd1-99f2-72b02b2602b3","owner":[],"postedDate":"February 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28688023,"name":"Biological sciences/Biophysics/Membrane structure and assembly"},{"id":28688024,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"}],"tags":[],"updatedAt":"2025-03-01T08:07:15+00:00","versionOfRecord":{"articleIdentity":"rs-3845625","link":"https://doi.org/10.1038/s41467-025-57152-7","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-02-28 05:00:00","publishedOnDateReadable":"February 28th, 2025"},"versionCreatedAt":"2024-02-12 02:13:59","video":"","vorDoi":"10.1038/s41467-025-57152-7","vorDoiUrl":"https://doi.org/10.1038/s41467-025-57152-7","workflowStages":[]},"version":"v1","identity":"rs-3845625","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3845625","identity":"rs-3845625","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}