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The Ig3 domain of MYPN mediates direct binding and bundling of filamentous actin (F\u2010actin), and several cardiomyopathy (CM)\u2010associated mutations cluster within this domain. To elucidate the molecular basis of MYPN\u2010actin interactions, we performed alanine\u2010scanning mutagenesis of conserved basic residues and evaluated CM\u2010linked variants of the Ig3 domain using co\u2010sedimentation assays. These mutations impaired actin binding and abolished bundling activity, despite preserving secondary structure in most cases. Circular dichroism revealed that the P961L variant adopts a more open, partially unfolded \u03b2\u2010sandwich conformation, distinct from the wild type (WT), which correlates with its aggregation and mislocalization in Drosophila cardiomyocytes. In vivo expression of full\u2010length WT and mutant MYPN in\n                    <jats:italic>Drosophila<\/jats:italic>\n                    muscle confirmed Z\u2010disc localization for WT, while R955W and P961L mutants formed aberrant clusters. Biochemical assays demonstrated that MYPN Ig3 promotes actin polymerization and bundling even under non\u2010polymerizing conditions, and its interaction with actin is electrostatically driven but calcium\u2010independent. Sedimentation equilibrium analysis confirmed that MYPN Ig3 functions as a monomer, suggesting that bundling arises from dual actin\u2010binding surfaces rather than from self\u2010oligomerization. These findings establish the Ig3 domain as a structurally robust, actin\u2010regulatory module and identify disruption of MYPN\u2013actin interactions as a pathogenic mechanism in CM. Our study provides the first direct evidence linking Ig3 domain mutations to impaired actin dynamics and sarcomere disorganization, offering mechanistic insight into MYPN\u2010associated cardiomyopathies.\n                  <\/jats:p>","DOI":"10.1002\/pro.70490","type":"journal-article","created":{"date-parts":[[2026,1,24]],"date-time":"2026-01-24T12:59:17Z","timestamp":1769259557000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Cardiomyopathy\u2010associated and basic residue mutations in myopalladin alter actin binding, bundling, and structural stability"],"prefix":"10.1002","volume":"35","author":[{"given":"Asha Rankoth","family":"Arachchige","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry Wichita State University  Wichita Kansas 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K.","family":"Kadarla","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry Wichita State University  Wichita Kansas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1574-5219","authenticated-orcid":false,"given":"Michal","family":"Zolkiewski","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics Kansas State University  Wichita Kansas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erika R.","family":"Geisbrecht","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics Kansas State University  Wichita Kansas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1004-7147","authenticated-orcid":false,"given":"Moriah R.","family":"Beck","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry Wichita State University 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