Arachchige, A.R., Tran, J., Zhao, Z., Dammann, H., Michaelis, A., Kadarla, V.K., Zolkiewski, M., Geisbrecht, E.R., Beck, M.R. (2026). Cardiomyopathy-associated and basic residue mutations in myopalladin alter actin binding, bundling, and structural stability.  Protein Sci. 35(2): e70490.

FBrf0264424

Research paper

Myopalladin (MYPN) is a striated muscle-specific protein essential for sarcomere integrity and actin filament organization. The Ig3 domain of MYPN mediates direct binding and bundling of filamentous actin (F-actin), and several cardiomyopathy (CM)-associated mutations cluster within this domain. To elucidate the molecular basis of MYPN-actin interactions, we performed alanine-scanning mutagenesis of conserved basic residues and evaluated CM-linked variants of the Ig3 domain using co-sedimentation 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 β-sandwich conformation, distinct from the wild type (WT), which correlates with its aggregation and mislocalization in Drosophila cardiomyocytes. In vivo expression of full-length WT and mutant MYPN in Drosophila muscle confirmed Z-disc 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-polymerizing conditions, and its interaction with actin is electrostatically driven but calcium-independent. Sedimentation equilibrium analysis confirmed that MYPN Ig3 functions as a monomer, suggesting that bundling arises from dual actin-binding surfaces rather than from self-oligomerization. These findings establish the Ig3 domain as a structurally robust, actin-regulatory module and identify disruption of MYPN-actin 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-associated cardiomyopathies.

PMC12831284 (PMC) (EuropePMC)