Discovery of a new evolutionarily conserved short linear F-actin binding motif

Discovery of a new evolutionarily conserved short linear F-actin binding 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 Article Discovery of a new evolutionarily conserved short linear F-actin binding motif Sabine Windhorst, Themistoklis Paraschiakos, Biao Yuan, Kostiantyn Sopelniak, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6464525/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Regulation of the actin cytoskeleton by actin binding proteins (ABPs) is essential for cellular homeostasis, and the mode of actin binding determines the activity of ABPs. Here, we discovered a novel “Short linear F-actin binding motif (SFM)” on the basis of the cryo-EM structure of the ITPKA-F-actin complex. We developed the computational pipeline SLiMFold, which identified 103 human SFM containing-proteins exhibiting diverse cellular functions. The SFM probably developed ex nihilo and remained conserved in eukaryotes, with a binding affinity to F-actin ranging from 13 to 89 µM. Furthermore, we uncovered the essential amino acids of this SFM for F-actin binding and affinity modulation. Together, the SFM seems to serve as a low affinity anchor to target proteins to F-actin, in order to connect the regulation of actin dynamics with broad cellular functions. These findings will shed new light on the role of a wide variety of proteins. Biological sciences/Biochemistry/Proteins Biological sciences/Computational biology and bioinformatics/Cellular signalling networks Biological sciences/Cell biology/Cytoskeleton/Actin Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Additional Declarations There is NO Competing Interest. Table 1 is available in the Supplementary Files section. Supplementary Files Tables.docx Tables D1292146312valreportfullP1ITPKAFactinEMD531339QGK.pdf Validation Report SupplementaryTables.docx Supplemetary Table ExtendedData.docx Extended Data Figures Cite Share Download PDF Status: Under Review 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-6464525","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":457196329,"identity":"3c1baf53-243c-4eb6-9349-9777f5d21d80","order_by":0,"name":"Sabine Windhorst","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYJCCAwxsB0A04wOGA2Au0VqYmQ2I1sIA1cImQZQW+QYew8MFZXfkzPvPH6vmOWPDwHe8Ab8WgwM8BodnnHtmLHMjme02z400BskzBKwxYODdcJi37XDiDAlmoJYPhxkMbiQQchhMC/9htmKeD/8ZDO4/IOCZAzAtDMlszDw3DgBtIaDD4DD/h8M85w4bS0gkG0vOOZPMI3mGkMPa25I/85QdlpPgP/jww5tjdnJ8xw8QsIYZjc9DQP0oGAWjYBSMAmIAAEA+SBopI5cmAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf","correspondingAuthor":true,"prefix":"","firstName":"Sabine","middleName":"","lastName":"Windhorst","suffix":""},{"id":457196330,"identity":"10a7ef64-7054-45cd-9532-28928c1670b5","order_by":1,"name":"Themistoklis Paraschiakos","email":"","orcid":"","institution":"Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Themistoklis","middleName":"","lastName":"Paraschiakos","suffix":""},{"id":457196331,"identity":"10f08ac9-549b-4c1b-9ecc-0c6d82a06273","order_by":2,"name":"Biao Yuan","email":"","orcid":"","institution":"Molecular Structural Biology, Helmholtz Center for Infection Research","correspondingAuthor":false,"prefix":"","firstName":"Biao","middleName":"","lastName":"Yuan","suffix":""},{"id":457196332,"identity":"ed9cfef3-0007-4b63-8270-8f361f16dca5","order_by":3,"name":"Kostiantyn Sopelniak","email":"","orcid":"","institution":"Institute for Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Kostiantyn","middleName":"","lastName":"Sopelniak","suffix":""},{"id":457196333,"identity":"42c24020-7918-4f6d-90c4-a9b33fb16fee","order_by":4,"name":"Michael Hecht-Bucher","email":"","orcid":"","institution":"Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Hecht-Bucher","suffix":""},{"id":457196334,"identity":"255bc737-a272-425c-9ea7-8e983e82c555","order_by":5,"name":"Lisa Simon","email":"","orcid":"","institution":"Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Lisa","middleName":"","lastName":"Simon","suffix":""},{"id":457196335,"identity":"9e68e4df-5712-47ef-8171-0541e3b73b8d","order_by":6,"name":"Ksenija Zonjic","email":"","orcid":"","institution":"Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Ksenija","middleName":"","lastName":"Zonjic","suffix":""},{"id":457196336,"identity":"89caa41e-0a74-4ea7-8039-d88908aa2286","order_by":7,"name":"Dominic Eggers","email":"","orcid":"","institution":"Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Dominic","middleName":"","lastName":"Eggers","suffix":""},{"id":457196337,"identity":"a152f0d0-cc84-468c-829a-8fccb05da320","order_by":8,"name":"Franziska Selle","email":"","orcid":"","institution":"Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Franziska","middleName":"","lastName":"Selle","suffix":""},{"id":457196338,"identity":"9a01d154-1fb6-481b-a61d-ff1a61b5a65d","order_by":9,"name":"Jing Li","email":"","orcid":"","institution":"Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Li","suffix":""},{"id":457196339,"identity":"6d175bb7-2821-4fbb-bb67-02337f707c80","order_by":10,"name":"Stefan Linder","email":"","orcid":"https://orcid.org/0000-0001-8226-2802","institution":"University Medical Center Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Linder","suffix":""},{"id":457196340,"identity":"4b1aa4fc-dc41-4c2f-afdc-1d43326a44a5","order_by":11,"name":"Thomas Marlovits","email":"","orcid":"","institution":"Institute of Microbial and Molecular Sciences, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Marlovits","suffix":""}],"badges":[],"createdAt":"2025-04-16 14:36:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6464525/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6464525/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82893415,"identity":"8e876977-88d2-48b6-94af-8d0983b8fe8e","added_by":"auto","created_at":"2025-05-16 12:23:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23228301,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural alignment of ITPKA\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eArg28-Ala49\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and Lifeact points towards a novel F-actin binding motif, prompting proteome-wide discovery using the SLiMFold pipeline. (a)\u003c/strong\u003e Structural superimposition of the ITPKA\u003csup\u003eArg28-Ala49\u003c/sup\u003e–actin complex (orange and red for the two actin subunits, cyan for ITPKA) with the Lifeact–actin structure (grey; PDB: 7AD9) shows near-identical actin conformations apart from a minor shift in the D-loop of the ITPKA-bound model. \u003cstrong\u003e(b)\u003c/strong\u003e Rotating the helices by 180° highlights the residues interacting with F-actin. Conserved side chains are represented as sticks and labeled P1, P4, P8, and P9. \u003cstrong\u003e(c) \u003c/strong\u003eDomain architecture of \u003cem\u003eS. cerevisiae\u003c/em\u003e ABP140 and \u003cem\u003eH. sapiens\u003c/em\u003e ITPKA. ABP140 comprises an N-terminal F-actin binding domain (ABD) and a C-terminal methyltransferase (MTase) domain. ITPKA features an N-terminal ABD, a central calmodulin-binding domain (CaMBD), and a C-terminal inositol 1,4,5-trisphosphate 3-kinase (IP3K) domain. Notably, the F-actin binding motifs, derived in this study, are located within intrinsically disordered regions, which is typical for short linear motifs (SLiMs). \u003cstrong\u003e(d)\u003c/strong\u003e Sequence comparison underscores structural and sequence conservation, conserved positions P1, P4, P8, and P9 are labeled in red. \u003cstrong\u003e(e)\u003c/strong\u003e SLiMFold pipeline overview. The SLiMFold pipeline started with the hypothesized SLiMs of Lifeact and ITPKA\u003csup\u003eArg28-Ala49\u003c/sup\u003e Sequence, on which basis a position-specific scoring matrix (PSSM) was calculated to identify motif hits from NCBI databases which were filtered by PSSM scores, IUPRED, ANCHOR, and PSIPRED. This yielded 539 hits in the first iteration. Thereafter, multiple sequence alignments (MSAs) were calculated locally using UniRef90 with modified jackhmmer filters, followed by multimer predictions on ColabFold. The predicted structures were analyzed by extracting AlphaFold2 scores, computing RMSD and angle metrics (θ, φ). These data were clustered to identify 59 new sequences that carry the suspected motif. \u003cstrong\u003e(f-i)\u003c/strong\u003e Detailed illustration of calculated metadata from (e).\u003cstrong\u003e (f) \u003c/strong\u003eScatterplot of predicted peptide–actin complexes plotting mean ipTM vs. RMSD relative to ITPKA\u003csup\u003eArg28-Ala49\u003c/sup\u003e. A red dotted line at ipTM=0.6 marks the threshold below which interactions are unreliable. \u003cstrong\u003e(g)\u003c/strong\u003e Helix orientation was assessed by comparing ∆φ and ∆θ angles in a polar coordinate system after superimposing each complex onto the ITPKA\u003csup\u003eArg28-Ala49\u003c/sup\u003e–actin reference. \u003cstrong\u003e(h)\u003c/strong\u003e A 3D plot of ∆φ, ∆θ, and helix polarity (1 = same direction, −1 = opposite) maps each predicted peptide, with data points colored by log(1+RMSD). Angles near zero indicate an orientation similar to that of ITPKA\u003csup\u003eArg28-Ala49\u003c/sup\u003e.\u003cstrong\u003e (i)\u003c/strong\u003e HDBSCAN clustering of these data points groups peptides based on orientation and RMSD. Outliers are assigned to Cluster –1, while Cluster 3 contains helices most closely matching ITPKA\u003csup\u003eArg28-Ala49\u003c/sup\u003e. \u003cstrong\u003e(j)\u003c/strong\u003e Sequence logo depicting positional conservation among the 59 identified peptides confirms robust conservation at positions P1, P4, P8, and P9, strongest at P1 and P8.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6464525/v1/941909d07a63c9b27cf7d242.png"},{"id":82892004,"identity":"8b8948f1-6330-4494-a813-d6df009b5a63","added_by":"auto","created_at":"2025-05-16 12:15:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":59702866,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation and functional analysis of the novel short linear F-actin binding motif (SFM) by cellular F-actin colocalization. (a)\u003c/strong\u003e Five peptides identified by the SLiMFold pipeline were aligned with the sequences of ITPKA and Lifeact; the conserved positions P1, P4, P8, P9 are highlighted in red. To probe their functional significance, the peptides were cloned into an pEGFP vector, and alanine mutations were introduced at P1 and P8. \u003cstrong\u003e(b)\u003c/strong\u003e Primary human macrophages were transfected with the vectors coding for the EGFP-tagged peptides and stained with phalloidin-568 to visualize F-actin (red). Representative cell images are shown including zoomed- into one podosome substructure, scale bars: 10 and 5 µm, respectively. Colocalization is evident when EGFP-tagged peptides (green) overlap with phalloidin-stained actin (red). Right panels: Imaris 3D reconstructions and Poji radial profiles confirm varying degrees of peptide–actin colocalization. Shown are Poji radial profiles of fluorescence intensities, at a z plane of highest F-actin intensity, with the mean values of F-actin and respective peptide ± standard deviation of at least 3 cells, and 240 podosomes per transfected peptide. Poji radial profiles were normalized to set the intensity values from 0% to 100%.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6464525/v1/5c521d57b4466231af331285.png"},{"id":82891989,"identity":"40a23c36-1fc3-4baf-90a1-4e49e94dcfaf","added_by":"auto","created_at":"2025-05-16 12:15:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17127744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetermination of SFM-binding affinities to F-actin, and identification of affinity-modulating amino acids.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Six further peptides identified by the SLiMFold pipeline were aligned with the sequences of ITPKA and Lifeact, and the conserved positions P1, P4, P8, and P9 highlighting in red. The peptides were expressed in bacteria, and after purification F-actin pull-downs were performed. Binding of the peptides were analyzed by Western blotting, and to calculate relative dissociation constants (K\u003csub\u003eD\u003c/sub\u003e values), the F-actin concentrations were plotted against normalized band intensities. The right panel provides the K\u003csub\u003eD\u003c/sub\u003e-values for all peptides tested. \u003cstrong\u003e(c)\u003c/strong\u003e In order to identify the amino acids modulating the affinity of the peptides to F-actin, their frequency was analyzed by generating a position specific matrix (PSFM) using MSA from CEFIP, USP54, ITPKA, PPP1R9A, Lifeact, ARHGEF11, FGD4, DIXDC1. Y-axis represents positively charged (+), negatively charged (-), polar (P), unique (U), non-polar (NP) and aromatic (A) residues, and X-axis the position inside the SFM. This matrix, color-coded by √(amino acid frequency), pinpointed less (white) and more frequent (red) residues. \u003cstrong\u003e(d)\u003c/strong\u003e SHROOM3 and USP54 wild-type (WT) sequences and corresponding mutants, including the key residues P1, P4, P8, P9. M2 features mutations N-terminal to the motif, M3 contains mutations within the motif, and M4 includes mutations C-terminal to the motif. M1 incorporates all mutations (N-terminal, within, and C-terminal). Newly mutated positions are labeled in red. \u003cstrong\u003e(e)\u003c/strong\u003e Quantified relative band intensities from SHROOM3 and USP54 pulldown assays, normalized and averaged across replicates, highlighting differences in binding affinities between WT and mutant peptides. (f) For USP54 M1 addiotinally the affinity to F-actin was determined. Statistical significance is denoted by asterisks: \u003cem\u003e*p \u0026lt; 0.05\u003c/em\u003e;\u0026nbsp; \u003cem\u003e**p \u0026lt; 0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6464525/v1/42af087ef07d4d2eb1be3f7e.png"},{"id":82893416,"identity":"10e9781f-7209-479f-8a68-1eb891ad712d","added_by":"auto","created_at":"2025-05-16 12:23:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20079141,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvolutionary analysis of validated SFM-containing proteins. (a)\u003c/strong\u003eSequence logos of SFMs from representative organism classes (e.g., \u003cem\u003eActinopteri, Coelacanthiformes\u003c/em\u003e,\u003cem\u003e Amphibia\u003c/em\u003e,\u003cem\u003e Mammalia\u003c/em\u003e,\u003cem\u003e Lepidosauria\u003c/em\u003e,\u003cem\u003e Testudines\u003c/em\u003e,\u003cem\u003e Aves\u003c/em\u003e, and\u003cem\u003e Crocodylia\u003c/em\u003e). Key motif positions P1, P4, P8, and P9 are labeled. Larger letters indicate higher residue conservation, reinforcing the importance of these positions for F-actin binding. \u003cstrong\u003e(b)\u003c/strong\u003e A dN/dS ratio analysis of all validated SFM-containing peptides is plotted ± SEM, with amino acid position on the x-axis and the dN/dS ratio on the y-axis. The conserved SFM residues (P1, P4, P8, P9) are annotated to highlight their selective pressures. Notably, these positions exhibit lower dN/dS values, suggesting strong purifying selection. \u003cstrong\u003e(c)\u003c/strong\u003e Phylogenetic tree illustrating the distribution of validated SFM-containing proteins across various species. Symbols mark the most recent common ancestor (MRCA) \u003cem\u003eSaccharomyces\u003c/em\u003e (ABP140), \u003cem\u003eBilateria\u003c/em\u003e (SHROOM3), \u003cem\u003eVertebrata\u003c/em\u003e (CGNL1, DIXDC1, FGD4, PPP1R9A), \u003cem\u003eEuteleostomi\u003c/em\u003e (ARHGEF11, ITPKA), and \u003cem\u003eGnathostomata\u003c/em\u003e (CEFI, DENND1C, ESPLN, FGD1, FGD4, USP54).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6464525/v1/1e80cbde1173a2d81bfb8fcc.png"},{"id":82891991,"identity":"0443b099-8f43-4b04-90aa-4c67199fa992","added_by":"auto","created_at":"2025-05-16 12:15:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16976229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIterative SLiMFold pipeline expansion and functional classification of newly identified SFMf‐containing proteins. (a)\u003c/strong\u003e Scheme of three iterative SLiMFold rounds (for detailed description, see Figure 3), including \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein cellulo\u003c/em\u003e validation, yielding 124 peptide-sequences from 103 different genes. \u003cstrong\u003e(b)\u003c/strong\u003e Cartoon representation of a eukaryotic cell highlighting the functional categories of new SFM‐bearing proteins. SFM candidates are grouped according to their principal roles and subcellular localizations, encompassing direct actin modulators, actin regulators, other signaling proteins and enzymes, nuclear structure and RNA processing, chromatin modification and DNA repair, microtubule‐associated factors, cilia, and cell‐junction‐associated proteins. Proteins for which F-actin binding has been attributed to the SFM (e.g., validated by truncation studies) are marked with an asterisk (*), whereas proteins that bind F-actin but lack direct evidence linking the interaction specifically to the SFM are marked with a dagger (†). The cartoon was created with BioRender.com.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6464525/v1/26cf13b5c837fd9288dcc551.png"},{"id":82891984,"identity":"14781b0b-e251-479f-918c-da92747d46c9","added_by":"auto","created_at":"2025-05-16 12:15:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":80161,"visible":true,"origin":"","legend":"Tables","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6464525/v1/a7eea2c8ee8ee032aa3a01e0.docx"},{"id":82891988,"identity":"5acc1933-a144-4098-b5ec-f7ea9bb8e132","added_by":"auto","created_at":"2025-05-16 12:15:49","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1984097,"visible":true,"origin":"","legend":"Validation Report","description":"","filename":"D1292146312valreportfullP1ITPKAFactinEMD531339QGK.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6464525/v1/a68c4f40521e562510979222.pdf"},{"id":82891985,"identity":"49e2e455-cc4a-4257-82a1-4536509b68cb","added_by":"auto","created_at":"2025-05-16 12:15:49","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":47470,"visible":true,"origin":"","legend":"Supplemetary Table","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6464525/v1/78483f5a566d80b1016696e6.docx"},{"id":82892007,"identity":"3fbcc242-2b7a-4af7-83c8-28a41fda445b","added_by":"auto","created_at":"2025-05-16 12:15:50","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12346766,"visible":true,"origin":"","legend":"Extended Data Figures","description":"","filename":"ExtendedData.docx","url":"https://assets-eu.researchsquare.com/files/rs-6464525/v1/01892daf31a57d7bc0703fa0.docx"}],"financialInterests":"\u003cp\u003eThere is \u003cstrong\u003eNO\u003c/strong\u003e Competing Interest.\u003c/p\u003e\n\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e","formattedTitle":"Discovery of a new evolutionarily conserved short linear F-actin binding motif","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":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6464525/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6464525/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRegulation of the actin cytoskeleton by actin binding proteins (ABPs) is essential for cellular homeostasis, and the mode of actin binding determines the activity of ABPs. Here, we discovered a novel \u0026ldquo;Short linear F-actin binding motif (SFM)\u0026rdquo; on the basis of the cryo-EM structure of the ITPKA-F-actin complex. We developed the computational pipeline SLiMFold, which identified 103 human SFM containing-proteins exhibiting diverse cellular functions. The SFM probably developed \u003cem\u003eex nihilo\u003c/em\u003e and remained conserved in eukaryotes, with a binding affinity to F-actin ranging from 13 to 89 \u0026micro;M. Furthermore, we uncovered the essential amino acids of this SFM for F-actin binding and affinity modulation. Together, the SFM seems to serve as a low affinity anchor to target proteins to F-actin, in order to connect the regulation of actin dynamics with broad cellular functions. These findings will shed new light on the role of a wide variety of proteins.\u003c/p\u003e","manuscriptTitle":"Discovery of a new evolutionarily conserved short linear F-actin binding motif","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 12:15:44","doi":"10.21203/rs.3.rs-6464525/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-cell-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ncb","sideBox":"Learn more about [Nature Cell Biology](http://www.nature.com/ncb/)","snPcode":"","submissionUrl":"","title":"Nature Cell Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e6ed7abc-6cad-4502-98c8-81e5a496bd9e","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":48592596,"name":"Biological sciences/Biochemistry/Proteins"},{"id":48592597,"name":"Biological sciences/Computational biology and bioinformatics/Cellular signalling networks"},{"id":48592598,"name":"Biological sciences/Cell biology/Cytoskeleton/Actin"},{"id":48592599,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"}],"tags":[],"updatedAt":"2026-05-05T10:17:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-16 12:15:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6464525","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6464525","identity":"rs-6464525","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}