Year: 2026
Type: article
Abstract: Interdomain linkers in multidomain proteins influence spatial arrangement, flexibility, and cooperative binding, yet their functional roles remain underexplored. Palladin, an actin scaffold protein essential for cytoskeletal organization, contains tandem immunoglobulin domains (Ig3-4) separated by an unusually long (∼41-residue), arginine-rich, intrinsically disordered linker. While Ig3 mediates direct F-actin binding, the adjacent Ig4 domain and linker enhance binding and bundling, suggesting active roles beyond passive connection. Using targeted mutagenesis, actin co-sedimentation, bundling assays, and SAXS, we show that linker length, charge, and residue patterning are critical for activity. Shortening or replacing the 41-residue linker with the native Ig4-Ig5 linker reduced binding and abolished bundling, while small internal deletions decreased affinity but unexpectedly increased bundling efficiency. Neutralizing basic residues eliminated activity, while scrambling the sequence impaired both functions-demonstrating that electrostatic tuning and residue order, not just net charge, are essential. SAXS revealed that the native linker supports a broad, dynamic interdomain ensemble, enabling multiple binding-competent geometries. Shortened linkers restricted this conformational space, locking domains in extended orientations incompatible with efficient actin engagement. These results establish the Ig3-Ig4 linker as a finely tuned structural and electrostatic module that coordinates domain flexibility and filament crosslinking. More broadly, these findings highlight long, intrinsically disordered linkers as active determinants of cytoskeletal scaffold function, providing a general mechanism for modulating multivalent interactions in actin-binding proteins.
Source: Biochemical Journal
DOI: 10.1042/bcj20250187
Institution: Wichita State University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Funders: U.S. Department of Energy, Kansas IDeA Network of Biomedical Research Excellence, Instituto de Física de Cantabria, National Institutes of Health, Office of Science, Horizon 2020 Framework Programme, Institut National de Physique Nucléaire et de Physique des Particules, National Institute of General Medical Sciences, Basic Energy Sciences, Biological and Environmental Research, SLAC National Accelerator Laboratory
Awards: G1229526393, G1286251239, G1982653369, G2371548206, G262532246, G2853296776, G2921881625, G303061748, G315621713, G3503899968, G4235786860, G4581610328, G495301720, G498139845, Probing Actin Filament Assembly, Structure, and Dynamics by Palladin, G5438923238, G5570207115, G6550344761, G7248514903, G7283024673, G7285114270, G7396808078, G7863156295, G8382000481, G8390282621, G8858395165, G8925327636, G9000150948, Kansas IDeA Network of Biomedical Research Excellence 5, A Synchrotron Radiation Structural Biology Resource, G911061179, G969889393
OpenAlex ID: W7124829350