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The resulting fusion, EWS::FLI1, directly interacts with and engages in a dynamic interplay with EWS that drives tumorigenesis and regulates the function of both proteins. While EWS<jats:sup>LCD<\/jats:sup> is known to promote self\u2010association, the role of the RNA\u2010binding domains (RBDs) of EWS, which include arginine\u2013glycine\u2013glycine (RGG) repeat regions and a structured RNA\u2010recognition motif (RRM), remains less well understood. Here, we investigate the interplay between EWS<jats:sup>LCD<\/jats:sup> and RBDs using biomolecular condensation assays, microscopy, nuclear magnetic resonance (NMR) spectroscopy, and molecular simulations. Our studies reveal that RBDs differentially influence EWS<jats:sup>LCD<\/jats:sup> condensate formation and suggest that electrostatics and polypeptide\u2010chain length likely contribute to this interaction. NMR spectroscopy and molecular dynamics simulations further demonstrate that EWS<jats:sup>LCD<\/jats:sup> and the central RNA\u2010binding region, comprising the RRM and RGG2 domains, engage in transient, non\u2010specific interactions that are broadly distributed across both regions and involve diverse residue types. Specifically, tyrosine, polar residues, and proline within EWS<jats:sup>LCD<\/jats:sup> preferentially interact with arginine, glycine, and proline residues in the RBD. Atomistic simulations of EWS confirm that the full\u2010length protein exhibits a similar interaction profile with conserved chemical specificity, supporting a model in which a network of weak, distributed interdomain contacts underlies EWS self\u2010association. Together, these findings provide molecular insight into the mechanisms of EWS condensate formation and lay the groundwork for understanding how interdomain interactions regulate EWS and EWS::FLI1 function.<\/jats:p>","DOI":"10.1002\/pro.70316","type":"journal-article","created":{"date-parts":[[2025,9,22]],"date-time":"2025-09-22T12:30:41Z","timestamp":1758544241000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Molecular basis of <scp>EWS<\/scp> interdomain self\u2010association and its role in condensate formation"],"prefix":"10.1002","volume":"34","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5908-0912","authenticated-orcid":false,"given":"Erich J.","family":"Sohn","sequence":"first","affiliation":[{"name":"Greehey Children's Cancer Research Institute The University of Texas Health Science Center at San Antonio  San Antonio Texas USA"},{"name":"Department of Biochemistry and Structural Biology The 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