Conformational Dynamics and Membrane Insertion Mechanism of B4GALNT1 in Ganglioside Synthesis

Conformational Dynamics and Membrane Insertion Mechanism of B4GALNT1 in Ganglioside Synthesis | 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 Conformational Dynamics and Membrane Insertion Mechanism of B4GALNT1 in Ganglioside Synthesis Janet Deane, Jack Welland, Henry Barrow, Phillip Stansfeld This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5881806/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Glycosphingolipids (GSLs) are crucial membrane components involved in essential cellular pathways. Complex GSLs, known as gangliosides, are synthesised in the ER/Golgi by a series of glycosyltransferase enzymes. Imbalances in GSL metabolism can lead to severe diseases, often affecting the nervous system. B4GALNT1 is a key enzyme in the ganglioside synthetic pathway, synthesising complex gangliosides including GM2 and GD2 from GM3 and GD3. These products are precursors to the major brain gangliosides. Loss of B4GALNT1 function causes hereditary spastic paraplegia 26 (HSP26), while its overexpression is linked to cancers including childhood neuroblastoma. Here, we present crystal structures of the homodimeric B4GALNT1 enzyme demonstrating conformational changes upon binding of donor substrate ligands and product. These structures support a catalytic mechanism that involves dynamic remodelling of the substrate binding site during catalysis. We also demonstrate that processing of lipid substrates by B4GALNT1 is severely compromised when surface loops flanking the active site are mutated from hydrophobic residues to polar. Molecular dynamics simulations support that these loops can insert into the lipid bilayer explaining how B4GALNT1 accesses and processes lipid substrates. By combining structure prediction and molecular simulations we propose that this mechanism of dynamic membrane insertion is exploited by other, structurally distinct GSL synthesising enzymes. Biological sciences/Biochemistry/Structural biology/Molecular modelling Biological sciences/Biochemistry/Enzymes/Transferases Biological sciences/Biochemistry/Structural biology/X-ray crystallography Biological sciences/Biochemistry/Lipids/Sphingolipids Full Text Additional Declarations There is NO Competing Interest. Supplementary Files 250116SupplementaryInformation.pdf Supplementary Information B4GAAMDFinal.mp4 Atomistic simulation of B4GALNT1 with GM3 Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 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-5881806","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":413852234,"identity":"35f2b03c-ceb2-446c-8698-a9a08f86ccc4","order_by":0,"name":"Janet 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Complex GSLs, known as gangliosides, are synthesised in the ER/Golgi by a series of glycosyltransferase enzymes. Imbalances in GSL metabolism can lead to severe diseases, often affecting the nervous system. B4GALNT1 is a key enzyme in the ganglioside synthetic pathway, synthesising complex gangliosides including GM2 and GD2 from GM3 and GD3. These products are precursors to the major brain gangliosides. Loss of B4GALNT1 function causes hereditary spastic paraplegia 26 (HSP26), while its overexpression is linked to cancers including childhood neuroblastoma. Here, we present crystal structures of the homodimeric B4GALNT1 enzyme demonstrating conformational changes upon binding of donor substrate ligands and product. These structures support a catalytic mechanism that involves dynamic remodelling of the substrate binding site during catalysis. We also demonstrate that processing of lipid substrates by B4GALNT1 is severely compromised when surface loops flanking the active site are mutated from hydrophobic residues to polar. Molecular dynamics simulations support that these loops can insert into the lipid bilayer explaining how B4GALNT1 accesses and processes lipid substrates. By combining structure prediction and molecular simulations we propose that this mechanism of dynamic membrane insertion is exploited by other, structurally distinct GSL synthesising enzymes.","manuscriptTitle":"Conformational Dynamics and Membrane Insertion Mechanism of B4GALNT1 in Ganglioside Synthesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-12 08:22:27","doi":"10.21203/rs.3.rs-5881806/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":"7d072152-64f2-4588-954d-2bc03d50957c","owner":[],"postedDate":"February 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44130908,"name":"Biological sciences/Biochemistry/Structural biology/Molecular modelling"},{"id":44130909,"name":"Biological sciences/Biochemistry/Enzymes/Transferases"},{"id":44130910,"name":"Biological sciences/Biochemistry/Structural biology/X-ray crystallography"},{"id":44130911,"name":"Biological sciences/Biochemistry/Lipids/Sphingolipids"}],"tags":[],"updatedAt":"2025-07-02T07:56:48+00:00","versionOfRecord":{"articleIdentity":"rs-5881806","link":"https://doi.org/10.1038/s41467-025-60593-9","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-07-01 04:00:00","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-02-12 08:22:27","video":"","vorDoi":"10.1038/s41467-025-60593-9","vorDoiUrl":"https://doi.org/10.1038/s41467-025-60593-9","workflowStages":[]},"version":"v1","identity":"rs-5881806","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5881806","identity":"rs-5881806","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}