Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments

The release of inorganic phosphate (P i ) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. However, the molecular mechanisms underlying P i release from both the core and barbed end of actin filaments remain unclear. Here, we combine cryo-EM with molecular dynamics simulations and in vitro reconstitution to demonstrate how actin releases P i through a ‘molecular backdoor’. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and only opens transiently through concerted backbone movements and rotameric rearrangements of residues close to the nucleotide binding pocket. This mechanism explains why P i escapes rapidly from the filament end and yet slowly from internal actin subunits. In an actin variant associated with nemaline myopathy, the backdoor is predominantly open in filament-core subunits, resulting in greatly accelerated P i release after polymerization and filaments with drastically shortened ADP-P i caps. This demonstrates that the P i release rate from F-actin is controlled by steric hindrance through the backdoor rather than by the disruption of the ionic bond between P i and Mg 2+ at the nucleotide-binding site. Our results provide the molecular basis for P i release from actin and exemplify how a single, disease-linked point mutation distorts the nucleotide state distribution and atomic structure of the actin filament.