Structural dynamics of mixed-subunit CaMKIIα/β heterododecamers filmed by high-speed AFM

Structural dynamics of mixed-subunit CaMKIIα/β heterododecamers filmed by high-speed AFM | 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 dynamics of mixed-subunit CaMKIIα/β heterododecamers filmed by high-speed AFM Mikihiro Shibata, Keisuke Matsushima, Takashi Sumikama, Taisei Suzuki, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6519178/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract CaMKII predominantly assembles into a 12-meric ring assembly, primarily consisting of CaMKIIα and CaMKIIβ variants in the brain. Previous biochemical studies have reported varying ratios of these CaMKII variants across different brain regions and developmental stages. However, direct evidence for the formation of CaMKIIα/β heterooligomers within a 12-meric ring assembly has been lacking at the single-molecule level. Here, we employed high-speed atomic force microscopy to visualize the conformational dynamics of forebrain-mimicked CaMKIIα/β at a 3:1 ratio. Our findings revealed that the CaMKIIα and CaMKIIβ subunits are intermixed within the 12-meric ring assembly, with more than 83% probability that CaMKIIβ subunits adjacent to one another. Furthermore, in the activated state, CaMKIIα/β heterooligomers form a stable kinase domain complex via interactions between adjacent CaMKIIβ subunits, resulting in a long-lasting structure with an exposed target binding site. Collectively, our observations provide insights into the structural role of CaMKIIβ subunits within the CaMKIIα/β heterododecamer. Biological sciences/Biophysics/Single-molecule biophysics Biological sciences/Neuroscience/Molecular neuroscience Biological sciences/Neuroscience/Synaptic plasticity/Long-term potentiation Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Sup.pdf Supplementary information SupMovie1CaMKIIBBasalVer2.mp4 HS-AFM videos of three representative CaMKIIβ homooligomers on a P[5]A+-modified mica surface. SupMovie2CaMKIIBBOSVer2.mp4 HS-AFM videos of three representative CaMKIIβ homooligomers with 50 μM bosutinib on a P[5]A+-modified mica surface. SupMovie3CaMKIIHeteroBasalVer2.mp4 HS-AFM videos of three representative CaMKIIα/β 3:1 heterooligomers on a P[5]A+-modified mica surface. SupMovie4CaMKIIGFPBBasalVer2.mp4 HS-AFM videos of three representative CaMKIIα/GFP-CaMKIIβ 3:1 heterooligomers on a P[5]A+-modified mica surface. SupMovie5CaMKIIBCaMVer2.mp4 HS-AFM videos of three representative CaMKIIβ homooligomers treated with Ca 2+ /CaM (1 mM Ca 2+ and 800 nM CaM) on a P[5]A+-modified mica surface. SupMovie6CaMKIIHeteroCaMVer2.mp4 HS-AFM videos of three representative CaMKIIα/β 3:1 heterooligomers treated with Ca 2+ /CaM (1 mM Ca 2+ and 800 nM CaM) on a P[5]A+-modified mica surface. SupMovie7CaMKIIBCaMpT287Ver2.mp4 HS-AFM videos of three representative CaMKIIβ homooligomers treated with Ca 2+ /CaM and ATP (1 mM Ca 2+ , 800 nM CaM, and 1 mM ATP) on a P[5]A+-modified mica surface. SupMovie8CaMKIIHeteropT286Ver2.mp4 HS-AFM videos of three representative CaMKIIα/β 3:1 heterooligomers treated with Ca 2+ /CaM and ATP (1 mM Ca 2+ , 800 nM CaM, and 1 mM ATP) on a P[5]A+-modified mica surface. SupMovie9CaMKIIBpTFullVer2.mp4 HS-AFM videos of three representative CaMKIIβ homooligomers treated with EGTA (2 mM) and ATP (1 mM) after Ca 2+ /CaM/ATP stimulation (1 mM Ca 2+ , 800 nM CaM, and 1 mM ATP) on a P[5]A+-modified mica surfa SupMovie10CaMKIIHeteropTFullVer2.mp4 HS-AFM videos of three representative CaMKIIα/β 3:1 heterooligomers treated with EGTA (2 mM) and ATP (1 mM) after Ca 2+ /CaM/ATP stimulation (1 mM Ca 2+ , 800 nM CaM, and 1 mM ATP) on a P[5]A+-modified mi SupMovie11T2867ACaMDissociationCombineVer1.mp4 HS-AFM videos of CaMKIIαT286A and CaMKIIβT287A homooligomers treated with Ca 2+ /CaM (1 mM Ca 2+ and 800 nM CaM) on a P[5]A+-modified mica surface. Cite Share Download PDF Status: Published Journal Publication published 24 Dec, 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-6519178","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":454689607,"identity":"372aa703-ef4f-45eb-b9c4-8b90becafe50","order_by":0,"name":"Mikihiro 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AFM","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature 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Previous biochemical studies have reported varying ratios of these CaMKII variants across different brain regions and developmental stages. However, direct evidence for the formation of CaMKIIα/β heterooligomers within a 12-meric ring assembly has been lacking at the single-molecule level. Here, we employed high-speed atomic force microscopy to visualize the conformational dynamics of forebrain-mimicked CaMKIIα/β at a 3:1 ratio. Our findings revealed that the CaMKIIα and CaMKIIβ subunits are intermixed within the 12-meric ring assembly, with more than 83% probability that CaMKIIβ subunits adjacent to one another. Furthermore, in the activated state, CaMKIIα/β heterooligomers form a stable kinase domain complex via interactions between adjacent CaMKIIβ subunits, resulting in a long-lasting structure with an exposed target binding site. Collectively, our observations provide insights into the structural role of CaMKIIβ subunits within the CaMKIIα/β heterododecamer.","manuscriptTitle":"Structural dynamics of mixed-subunit CaMKIIα/β heterododecamers filmed by high-speed AFM","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-21 07:27:00","doi":"10.21203/rs.3.rs-6519178/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":"a71c4cfe-d8ab-411d-beb0-cfbde2a58318","owner":[],"postedDate":"May 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48345835,"name":"Biological sciences/Biophysics/Single-molecule biophysics"},{"id":48345836,"name":"Biological sciences/Neuroscience/Molecular neuroscience"},{"id":48345837,"name":"Biological sciences/Neuroscience/Synaptic plasticity/Long-term potentiation"}],"tags":[],"updatedAt":"2025-12-25T08:05:34+00:00","versionOfRecord":{"articleIdentity":"rs-6519178","link":"https://doi.org/10.1038/s41467-025-66527-9","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-24 05:00:00","publishedOnDateReadable":"December 24th, 2025"},"versionCreatedAt":"2025-05-21 07:27:00","video":"","vorDoi":"10.1038/s41467-025-66527-9","vorDoiUrl":"https://doi.org/10.1038/s41467-025-66527-9","workflowStages":[]},"version":"v1","identity":"rs-6519178","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6519178","identity":"rs-6519178","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}