Key determinants of VDAC-hexokinase I complex assembly revealed by a minimal vesicle-based interaction assay

Key determinants of VDAC-hexokinase I complex assembly revealed by a minimal vesicle-based interaction assay | 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 Key determinants of VDAC-hexokinase I complex assembly revealed by a minimal vesicle-based interaction assay Joost Holthuis, Milena Wessing, Isabelle Watrinet, Michael Timme, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8079261/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Binding of hexokinase HKI to mitochondrial voltage-dependent anion channels (VDACs) regulates the metabolic fate of glucose and promotes cell survival in hyperglycolytic tumors. Computer simulations indicated that complex assembly relies on intimate contacts between the N-terminal α-helix of HKI and a charged bilayer-facing glutamate on the outer channel wall. Protonation of this residue blocks complex formation in silico, explaining the release of HKI from mitochondria observed upon cytosolic acidification. To validate these findings, we here developed an in vitro assay for interrogating HKI binding to VDAC1 reconstituted in vesicles captured onto a functionalized surface. TIRF-based quantitative interaction studies with a fluorescent peptide comprising the N-terminal α-helix of HKI revealed a crucial role of the bilayer-facing glutamate in complex assembly and recapitulated an exquisite sensitivity of HKI-VDAC binding to fluctuations in pH. Our assay opens up important opportunities to investigate the impact of membrane environment on HKI-VDAC complex assembly and may benefit the development of therapeutics that target pathogenic imbalances in this process. Biological sciences/Biochemistry/Ion channels/Porins Biological sciences/Biophysics/Molecular biophysics/Supramolecular assembly Biological sciences/Biological techniques/Imaging/Fluorescence imaging Biological sciences/Biological techniques/Nanobiotechnology/Nanofabrication and nanopatterning Full Text Additional Declarations The authors declare no competing interests. Supplementary Files WessingSI.pdf Supplemental Information COMMSBIO2510937RS.pdf Reporting Summary Cite Share Download PDF Status: Posted 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. 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