Redefining PH Domain Function: An Active Allosteric Mechanism in ASAP1-Mediated Arf1 GTP Hydrolysis

Redefining PH Domain Function: An Active Allosteric Mechanism in ASAP1-Mediated Arf1 GTP Hydrolysis | 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 Redefining PH Domain Function: An Active Allosteric Mechanism in ASAP1-Mediated Arf1 GTP Hydrolysis Paul Randazzo, Olivier soubias, Samuel Foley, Xiaoying Jian, Rebekah Jackson, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6702895/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract GTPase-activating proteins (GAPs) are important regulators of small GTPases with a wide range of cellular functions; among these, ASAP1 stimulates GTP hydrolysis on Arf1 and is implicated in cancer progression. ASAP1 contains a Pleckstrin Homology (PH) domain critical for maximum hydrolysis of GTP bound to the small GTPase Arf. The prevailing view of PH domains is that they regulate proteins by passive mechanisms such as recruitment to the membrane surface. In sharp contrast to this model of regulation, our research reveals that the PH domain of ASAP1 actively contributes to Arf1 GTP hydrolysis. By combining NMR, molecular dynamics simulations, kinetic assays, and mutational analysis, we found that the PH domain directly interacts with Arf·GTP at the membrane, to drive conformational rearrangements of the GTP binding site. These structural changes establish an active state primed for GTP hydrolysis, facilitating charge stabilization which in turn, significantly enhances the catalytic rate of the GTPase reaction. Specifically, we identified key residues on both the PH domain and Arf responsible for this allosteric mechanism. Further, through mathematical modeling, we quantified the contribution of this newly discovered allosteric mechanism to ASAP1 GTPase-activating protein activity and found that it contributes equally to GTPase activation as membrane recruitment. The discovery that PH domains can directly affect nucleotide hydrolysis by a small GTPase has ramifications for the larger group of small GTPases, that include Ras and Rho proteins, that are regulated by proteins with PH domains, control diverse cellular functions and are oncoproteins. Biological sciences/Biochemistry/Enzyme mechanisms Biological sciences/Structural biology/NMR spectroscopy Biological sciences/Structural biology/Molecular modelling Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SUPPFINAL20250512.pdf Supplementary figures and methods Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 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-6702895","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":460469681,"identity":"262402e2-a0ae-4479-8344-2682d770db08","order_by":0,"name":"Paul 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