Molecular Basis for the Calcium-Dependent Activation of the Ribonuclease EndoU

Molecular Basis for the Calcium-Dependent Activation of the Ribonuclease EndoU | 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 Molecular Basis for the Calcium-Dependent Activation of the Ribonuclease EndoU Florian Malard, Kristen Dias, Margaux Baudy, Stéphane Thore, Brune Vialet, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4654759/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Apr, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Ribonucleases (RNases) are ubiquitous enzymes that process or degrade RNA, essential for cellular functions and immune responses. The EndoU-like superfamily includes endoribonucleases conserved across bacteria, eukaryotes, and certain viruses, with an ancient evolutionary link to the ribonuclease A-like superfamily. Both bacterial EndoU and animal RNase A share a similar fold and function independently of cofactors. In contrast, the eukaryotic EndoU catalytic domain requires divalent metal ions for catalysis, possibly due to an N-terminal extension near the catalytic core. In this study, we used biophysical and computational techniques along with in vitro assays to investigate the calcium-dependent activation of human EndoU. We determined the crystal structure of EndoU bound to calcium and found that calcium binding remote from the catalytic triad triggers water-mediated intramolecular signaling and structural changes, activating the enzyme through allostery. Calcium-binding involves residues from both the catalytic core and the N-terminal extension, indicating that the N-terminal extension interacts with the catalytic core to modulate activity in response to calcium. Our findings suggest that similar mechanisms may be present across all eukaryotic EndoUs, highlighting a unique evolutionary adaptation that connects endoribonuclease activity to cellular signaling in eukaryotes. Biological sciences/Structural biology/X-ray crystallography Biological sciences/Structural biology/NMR spectroscopy/Solution-state NMR Biological sciences/Biochemistry/Enzyme mechanisms Biological sciences/Biophysics/Molecular biophysics/Molecular conformation Biological sciences/Chemical biology/RNA EndoU calcium RNA allostery ribonuclease. Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Fig3noncroppedgels.pdf Non-cropped gel images Malardetal2024SI.pdf Cite Share Download PDF Status: Published Journal Publication published 01 Apr, 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-4654759","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":326006350,"identity":"c94ae12c-ac22-49ab-934e-f62a38d7a94d","order_by":0,"name":"Florian Malard","email":"","orcid":"","institution":"INSERM U1212","correspondingAuthor":false,"prefix":"","firstName":"Florian","middleName":"","lastName":"Malard","suffix":""},{"id":326006351,"identity":"d48fd5ea-8726-4c6f-ae19-4da88f48e284","order_by":1,"name":"Kristen Dias","email":"","orcid":"","institution":"University of California 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The EndoU-like superfamily includes endoribonucleases conserved across bacteria, eukaryotes, and certain viruses, with an ancient evolutionary link to the ribonuclease A-like superfamily. Both bacterial EndoU and animal RNase A share a similar fold and function independently of cofactors. In contrast, the eukaryotic EndoU catalytic domain requires divalent metal ions for catalysis, possibly due to an N-terminal extension near the catalytic\r\ncore. In this study, we used biophysical and computational techniques along with in vitro assays to\r\ninvestigate the calcium-dependent activation of human EndoU. We determined the crystal structure\r\nof EndoU bound to calcium and found that calcium binding remote from the catalytic triad triggers\r\nwater-mediated intramolecular signaling and structural changes, activating the enzyme through allostery. Calcium-binding involves residues from both the catalytic core and the N-terminal extension, indicating that the N-terminal extension interacts with the catalytic core to modulate activity in response to calcium. 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