Binding of Phosphate, Aluminum Fluoride, or Beryllium Fluoride to F-actin Inhibits Severing by Gelsolin

Abstract

Actin exhibits ATPase activity of unknown function that increases when monomers polymerize into filaments. Differences in the kinetics of ATP hydrolysis and the release of the hydrolysis products ADP and inorganic phosphate suggest that phosphate-rich domains exist in newly polymerized filaments. We examined whether the enrichment of phosphate on filamentous ADP-actin might modulate the severing activity of gelsolin, a protein previously shown to bind differently to ATP and ADP actin monomers. Binding of phosphate, or the phosphate analogs aluminum fluoride and beryllium fluoride, to actin filaments reduces their susceptibility to severing by gelsolin. The concentration and pH dependence of inhibition suggest that HPO 4 2؊ binding to actin filaments generates this resistant state. We also provide evidence for two different binding sites for beryllium fluoride on actin. Actin has been postulated to contain two P i binding sites. Our data suggest that they are sequentially occupied following ATP hydrolysis by HPO 4 2؊ which is subsequently titrated to H 2 PO 4 ؊ . We speculate that beryllium fluoride and aluminum fluoride bind to the HPO 4 2؊ binding site. The cellular consequences of this model of phosphate release are discussed.

Flightless I interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling

“…On the basis of the actin-filament-capping activity of gelsolin and its amino acid sequence ( Sun et al. , 1994 ; Allen et al. , 1996 ), we considered that the actin-capping function of FliI may be associated with GLD 1.…”

ATP competes with PIP2 for binding to gelsolin

“…These residues also comprise part of a region that previously had been determined to bind to PIP 2 [ 27 ]. Gelsolin is also sensitive to the type of nucleotide bound to actin; it severs filaments that contain ADP-actin but not ADP-Pi-actin units [ 46 ]. Accordingly, G4-G6 (residues 418–741) shows a preference for ADP-containing actin monomers while G1-G3 (residues 25–370) binds to ATP- and ADP-actin with comparable affinities [ 47 ].…”

Investigating a back door mechanism of actin phosphate release by steered molecular dynamics

“…21,22 Kinetic experiments suggest that subsequent titration of P i is required to allow release from actin. 9,23 In the living cell, the regulation of P i release by its protonation state is believed to act as a clock, altering in a time-dependent manner the stability and mechanical properties of the filament. 23 Earlier studies singled out actin's methylated His73 as a putative modifier of P i release.…”

“…9,23 In the living cell, the regulation of P i release by its protonation state is believed to act as a clock, altering in a time-dependent manner the stability and mechanical properties of the filament. 23 Earlier studies singled out actin's methylated His73 as a putative modifier of P i release. 15 In the F-actin filament structure, 24 the toxin phalloidin binds to His73 near the entrance of the back door pathway (Fig.…”