In Silico Structural Characterization of Arthrobacter Phage Alatato Immunity Cassette Reveals a Conserved Arginine-Mediated Repressor Motif

In Silico Structural Characterization of Arthrobacter Phage Alatato Immunity Cassette Reveals a Conserved Arginine-Mediated Repressor Motif | 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 research-highlight In Silico Structural Characterization of Arthrobacter Phage Alatato Immunity Cassette Reveals a Conserved Arginine-Mediated Repressor Motif Rakin Khan, Aashi Kothari This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9577902/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 Understanding the regulatory mechanisms of viral lysogeny requires precise characterization of repressor-operator interactions. In this study, we investigated the unmapped immunity cassette of Arthrobacter phage Alatato (Cluster FB) utilizing in silico structural modeling. We identified gp31 and gp33 as potential functional DNA-binding repressors, which are predicted to utilize a strictly conserved Helix-Turn-Helix (HTH) motif to engage the major groove of the operator DNA. Through structural mapping and computational mutagenesis via AlphaFold 3, we identified Arginine 51 (R51) as a candidate residue mediating electrostatic interactions with the DNA phosphate backbone; in silico substitution of R51 to Alanine (R51A) notably reduced the predicted structural confidence of the binding complex. Furthermore, structural alignment of Alatato gp31 with its homolog from the genetically divergent Cluster FA phage Bridgette (Pham 289627) yielded a 0.556 Å RMSD, demonstrating strong structural similarity of the R51 interface. Conversely, the Cluster AZ1 phage Adolin (Pham 291150) exhibited no predictive binding affinity (ipTM 0.22) for the Alatato operator sequence, supporting functional divergence between these repressor families. These findings highlight a highly conserved, Arginine-mediated electrostatic binding mechanism that appears to be structurally maintained across distinct viral clusters. AlphaFold 3 UCSF ChimeraX Arthrobacter phage immunity cassette Helix-Turn-Helix motif repressor-operator interaction protein folding Full Text Additional Declarations No competing interests reported. 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. 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-9577902","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"research-highlight","associatedPublications":[],"authors":[{"id":634879190,"identity":"88b21df5-a630-4e92-92d7-92696709193f","order_by":0,"name":"Rakin Khan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYBACgxtAgvGPjQGQYmNsIEaL4QyQloY0ErQYS4C1HCZBi5l087PHlTvOG8tHJLA9nEGMFhuZY+aGZ8/cNjO8kcBuuIEoLRIJZpINbLdtDHsOsEk+IMphEunfgFrOkaDFWCLHTLKx7YCZPHsDmyRRDjOckVMm2XAm2diAvbFNkijvG9xI3ybZUGFnOL+Z+ZhkDzFaEHoPEBcrSECeVA2jYBSMglEwcgAAc9syi16d5VoAAAAASUVORK5CYII=","orcid":"","institution":"King George V School","correspondingAuthor":true,"prefix":"","firstName":"Rakin","middleName":"","lastName":"Khan","suffix":""},{"id":634879191,"identity":"0bcad716-127c-463b-9da8-06f0a6d67d91","order_by":1,"name":"Aashi Kothari","email":"","orcid":"","institution":"King George V School","correspondingAuthor":false,"prefix":"","firstName":"Aashi","middleName":"","lastName":"Kothari","suffix":""}],"badges":[],"createdAt":"2026-04-30 13:38:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9577902/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9577902/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108805410,"identity":"7dae8b11-8f54-4075-80cb-c972da7673dd","added_by":"auto","created_at":"2026-05-08 15:25:53","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":507212,"visible":true,"origin":"","legend":"","description":"","filename":"ResearchPaperonStructuralCharacterizationofArthrobacterPhageAlatatoImmunityCassetteRevealsaConservedArginineMediatedRepressorMotifrevised2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9577902/v1_covered_e24e7080-fb70-4723-916d-9532261b8876.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"In Silico Structural Characterization of Arthrobacter Phage Alatato Immunity Cassette Reveals a Conserved Arginine-Mediated Repressor Motif","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"AlphaFold 3, UCSF ChimeraX, Arthrobacter phage, immunity cassette, Helix-Turn-Helix motif, repressor-operator interaction, protein folding","lastPublishedDoi":"10.21203/rs.3.rs-9577902/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9577902/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding the regulatory mechanisms of viral lysogeny requires precise characterization of repressor-operator interactions. In this study, we investigated the unmapped immunity cassette of Arthrobacter phage Alatato (Cluster FB) utilizing in silico structural modeling. We identified gp31 and gp33 as potential functional DNA-binding repressors, which are predicted to utilize a strictly conserved Helix-Turn-Helix (HTH) motif to engage the major groove of the operator DNA. Through structural mapping and computational mutagenesis via AlphaFold 3, we identified Arginine 51 (R51) as a candidate residue mediating electrostatic interactions with the DNA phosphate backbone; in silico substitution of R51 to Alanine (R51A) notably reduced the predicted structural confidence of the binding complex. Furthermore, structural alignment of Alatato gp31 with its homolog from the genetically divergent Cluster FA phage Bridgette (Pham 289627) yielded a 0.556 Å RMSD, demonstrating strong structural similarity of the R51 interface. Conversely, the Cluster AZ1 phage Adolin (Pham 291150) exhibited no predictive binding affinity (ipTM 0.22) for the Alatato operator sequence, supporting functional divergence between these repressor families. These findings highlight a highly conserved, Arginine-mediated electrostatic binding mechanism that appears to be structurally maintained across distinct viral clusters.\u003c/p\u003e","manuscriptTitle":"In Silico Structural Characterization of Arthrobacter Phage Alatato Immunity Cassette Reveals a Conserved Arginine-Mediated Repressor Motif","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-06 11:02:17","doi":"10.21203/rs.3.rs-9577902/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7f084cbe-b4a9-4f77-a68c-cf0a427a6044","owner":[],"postedDate":"May 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T11:02:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-06 11:02:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9577902","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9577902","identity":"rs-9577902","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}