Structural basis of thymosin-β4/profilin exchange leading to actin filament polymerization

Significance Thymosin-β4 (Tβ4) sequesters actin monomers to help maintain the high concentrations of unpolymerized actin in higher eukaryotic cells. Despite more than two decades of research investigating the Tβ4–actin interaction, the X-ray structure of the full-length Tβ4:actin complex remained unresolved. Here, we report two X-ray structures of Tβ4:actin complexes. The first structure reveals that Tβ4 has two helices that bind at the barbed and pointed faces of actin, whereas the second structure displays a more open actin nucleotide binding cleft and a disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, reveal how Tβ4 prevents monomeric actin from joining actin filaments but participates in the exchange of actin with profilin to ensure controlled actin polymerization.