Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

Abstract

The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as "polymerization" and "stiffness" sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments.ion-linkage | structural bioinformatics | persistence length | polyelectrolyte T he polymerization of the protein actin into double-stranded helical filaments powers many eukaryotic cell movements and provides cells with mechanical strength and integrity (1-4). Filament formation is favored when the total actin concentration exceeds the critical concentration (C c ) for assembly-defined as the monomer concentration at steady state for ATP-actin, or the dissociation constant for the reversible-equilibrium binding reaction of monomer binding to ADP-actin filament ends. Accordingly, the C c of ADP-actin is linked to the filament subunit interaction free energy such that lower C c values reflect greater thermodynamic stability (5).The effects of solution ionic conditions on the assembly and stability of actin filaments have been investigated for several decades (6-12). The actin C c and (monomer and filament) conformation depend on the nucleotide-associated divalent cation (Ca 2þ or Mg 2þ ) as well as the type and concentration of ions in solution (6, 7, 13-15), a behavior shared among characterized actins and their bacterial homologs (16). However, it is not firmly established if these salt effects on actin filament assembly and mechanics originate from nonspecific ion effects (e.g., electrostatic screening, counterion condensation, etc.) and/or specific ion binding interactions, potentially at discrete sites. Identification of saturable cation binding sites with different affinities favors specific and discrete binding sites on monomers (8-10, 17), but the location of these sites and their contributions to filament assembly and stiffness are unknown.Here we identify distinct cation-binding sites at subunit interfaces that regulate actin filament assembly and rigidity. Sit...

Site-specific cation release drives actin filament severing by vertebrate cofilin

“…Saccharomyces cerevisiae (herein referred to as yeast) actin lacks an acidic residue (Glu167 in subdomain 3) required to form the stiffness site and filaments display mechanical properties that are not influenced by cations (19). In contrast, cations have a strong effect on the stiffness of yeast actin filaments engineered with Glu167 at the stiffness site (A167E) (19,22).…”

“…Cations modulate actin filament structure and mechanical properties (19) and cofilin dissociates filament-associated cations (20), leading us to hypothesize that cation-binding interactions regulate filament severing by cofilin. Cations bind filaments at two discrete and specific sites positioned between adjacent subunits along the long-pitch helix of the filament (19,21).…”

“…Cations bind filaments at two discrete and specific sites positioned between adjacent subunits along the long-pitch helix of the filament (19,21). These cation binding sites are referred to as "polymerization" and "stiffness" sites based on their roles in filament assembly and mechanics, respectively.…”

“…These cation binding sites are referred to as "polymerization" and "stiffness" sites based on their roles in filament assembly and mechanics, respectively. These discrete sites bind both monovalent and divalent cations with a range of affinities (low millimolar for divalent and tens of millimolar for monovalent cations) (19,21) but are predominantly occupied by Mg 2+ and K + under physiological conditions. Here we demonstrate that cation release from the stiffness site plays a central role in filament severing by vertebrate cofilin, both in vitro and in cells.…”

“…Saccharomyces cerevisiae (herein referred to as yeast) actin lacks an acidic residue (Glu167 in subdomain 3) required to form the stiffness site and filaments display mechanical properties that are not influenced by cations (19). In contrast, cations have a strong effect on the stiffness of yeast actin filaments engineered with Glu167 at the stiffness site (A167E) (19,22).To investigate the structural basis of the filament stiffness change introduced by the A167E substitution, we solved structures of A167E yeast actin filaments in low and high [Mg 2+ ] conditions by electron cryomicroscopy. Although the subunit conformational heterogeneity in filaments is evidently high (23), comparison of the density maps reveals cation-dependent structural differences that may reflect a shift toward a more rigid conformation at high Mg 2+ concentrations (Fig.…”