Molecular Mechanisms of Inorganic-Phosphate Release from the Core and Barbed End of Actin Filaments

Wout Oosterheert*, Florian E. C. Blanc*, Ankit Roy, Alexander Belyy, Micaela Boiero Sanders, Oliver Hofnagel, Gerhard Hummer, Peter Bieling, Stefan Raunser, (* equal contribution)
Nature Structural & Molecular Biology, 2023

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We combine cryo-EM, enhanced sampling MD simulations, machine learning, and in vivo and in vitro reconstitution to reveal the mechanisms and pathways for Pi release from F-actin, both from filament interior and barbed end.

Abstract

The release of inorganic phosphate (Pi) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying Pi release from the core and barbed end of actin filaments remain unclear. Here, using human and bovine actin isoforms, we combine cryo-EM with molecular-dynamics simulations and in vitro reconstitution to demonstrate how actin releases Pi through a `molecular backdoor'. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and opens only transiently through concerted amino acid rearrangements. This explains why Pi escapes rapidly from the filament end but slowly from internal subunits. In a nemaline-myopathy-associated actin variant, the backdoor is predominantly open in filament-core subunits, resulting in accelerated Pi release and filaments with drastically shortened ADP-Pi caps. Our results provide the molecular basis for Pi release from actin and exemplify how a disease-linked mutation distorts the nucleotide-state distribution and atomic structure of the filament.