Structural Basis for the Assembly of Amyloid Fibrils by the Master Cell-Signaling Regulator Human Receptor-Interacting Protein Kinase 1

Structural Basis for the Assembly of Amyloid Fibrils by the Master Cell-Signaling Regulator Human Receptor-Interacting Protein Kinase 1 | 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 Basis for the Assembly of Amyloid Fibrils by the Master Cell-Signaling Regulator Human Receptor-Interacting Protein Kinase 1 Miguel Mompeán, Jorge Pedro López-Alonso, Iban Ubarretxena-Belandia, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5820237/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Oct, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Amyloid fibrils, typically associated with neurodegenerative diseases, also play critical roles as functional assemblies in biological processes. The RIP homotypic interaction motifs (RHIMs) in receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) are essential for necroptosis, orchestrating the formation of amyloid-like fibrils that assemble into necrosomes. These supramolecular complexes propagate cell death signals and activate effectors like MLKL. While the structures of human RIPK3 (hRIPK3) homomeric fibrils and RIPK1-RIPK3 heteromeric fibrils have been resolved, the atomic structure of human RIPK1 (hRIPK1) homomeric fibrils has remained elusive. Here, we present a high-resolution structure of hRIPK1 RHIM-mediated amyloid fibrils, determined using an integrative approach combining cryoprobe-detected solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy. The fibrils adopt an N-shaped amyloid fold consisting of three β-sheets stabilized by the conserved IQIG RHIM motif through hydrophobic interactions and hydrogen bonding. A key hydrogen bond between N545 and G542 closes the β2-β3 loop, resulting in denser side-chain packing compared to hRIPK3 homomeric fibrils. This structural feature likely contributes to the compact architecture of hRIPK1 fibrils, in contrast to the more relaxed S-shaped fold observed in hRIPK3. These findings provide structural insights into how hRIPK1 homomeric fibrils nucleate hRIPK3 recruitment and fibrillization during necroptosis, offering broader perspectives on the molecular principles governing RHIM-mediated amyloid assembly and functional amyloids. Biological sciences/Structural biology/NMR spectroscopy/Solid-state NMR Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Biophysics Full Text Additional Declarations There is NO Competing Interest. Supplementary Files TableS1.xlsx Table S1 TableS2.xlsx Table S2 TableS3.xlsx Table S3 TableS4.xlsx Table S4 hRIPK1Polonio20250112SupportingInformation.pdf Supporting Information Cite Share Download PDF Status: Published Journal Publication published 30 Oct, 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-5820237","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":405241153,"identity":"4c047e9d-5216-41ba-8607-eb246deb8e50","order_by":0,"name":"Miguel 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The RIP homotypic interaction motifs (RHIMs) in receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) are essential for necroptosis, orchestrating the formation of amyloid-like fibrils that assemble into necrosomes. These supramolecular complexes propagate cell death signals and activate effectors like MLKL. While the structures of human RIPK3 (hRIPK3) homomeric fibrils and RIPK1-RIPK3 heteromeric fibrils have been resolved, the atomic structure of human RIPK1 (hRIPK1) homomeric fibrils has remained elusive.\r\n \r\nHere, we present a high-resolution structure of hRIPK1 RHIM-mediated amyloid fibrils, determined using an integrative approach combining cryoprobe-detected solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy. The fibrils adopt an N-shaped amyloid fold consisting of three β-sheets stabilized by the conserved IQIG RHIM motif through hydrophobic interactions and hydrogen bonding. A key hydrogen bond between N545 and G542 closes the β2-β3 loop, resulting in denser side-chain packing compared to hRIPK3 homomeric fibrils. This structural feature likely contributes to the compact architecture of hRIPK1 fibrils, in contrast to the more relaxed S-shaped fold observed in hRIPK3.\r\n \r\nThese findings provide structural insights into how hRIPK1 homomeric fibrils nucleate hRIPK3 recruitment and fibrillization during necroptosis, offering broader perspectives on the molecular principles governing RHIM-mediated amyloid assembly and functional amyloids.","manuscriptTitle":"Structural Basis for the Assembly of Amyloid Fibrils by the Master Cell-Signaling Regulator Human Receptor-Interacting Protein Kinase 1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-24 05:29:57","doi":"10.21203/rs.3.rs-5820237/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":"b6259c7c-8e7f-45a5-ae8e-380506b308b6","owner":[],"postedDate":"January 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43208124,"name":"Biological sciences/Structural biology/NMR spectroscopy/Solid-state NMR"},{"id":43208125,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":43208126,"name":"Biological sciences/Biophysics"}],"tags":[],"updatedAt":"2025-10-31T07:09:19+00:00","versionOfRecord":{"articleIdentity":"rs-5820237","link":"https://doi.org/10.1038/s41467-025-64621-6","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-10-30 04:00:00","publishedOnDateReadable":"October 30th, 2025"},"versionCreatedAt":"2025-01-24 05:29:57","video":"","vorDoi":"10.1038/s41467-025-64621-6","vorDoiUrl":"https://doi.org/10.1038/s41467-025-64621-6","workflowStages":[]},"version":"v1","identity":"rs-5820237","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5820237","identity":"rs-5820237","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}