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

Abstract

AbstractThe release of inorganic phosphate (Pi) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. However, the molecular mechanisms underlying Pirelease from both the core and barbed end of actin filaments remain unclear. Here, we combine cryo-EM with molecular dynamics simulations andin vitroreconstitution to demonstrate how actin releases Pithrough 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 Piescapes 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 Pirelease after polymerization and filaments with drastically shortened ADP-Picaps. This demonstrates that the Pirelease rate from F-actin is controlled by steric hindrance through the backdoor rather than by the disruption of the ionic bond between Piand Mg2+at the nucleotide-binding site. Our results provide the molecular basis for Pirelease from actin and exemplify how a single, disease-linked point mutation distorts the nucleotide state distribution and atomic structure of the actin filament.