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

️Sun Jan 01 2012

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

Hyeran Kang et al. Proc Natl Acad Sci U S A. 2012.

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Fig. 1.**
Specific cation binding drives actin polymerization. Linear fits of the activity coefficient (γ) corrected cation concentration dependence of K_polym (rabbit skeletal muscle actin, 5% pyrene labeled) yields slopes of 1.18 ± 0.02, 0.46 ± 0.10, 0.91 ± 0.09, and 0.83 ± 0.02, for K⁺, Na⁺, Mg²⁺, or Ca²⁺, respectively. Uncertainty bars represent the standard error (SEM).

**Fig. 2.**
Cation binding stiffens actin filaments. Bending persistence length (L_p) of actin filaments (rabbit skeletal muscle actin, Alexa 488 labeled) in K⁺, Na⁺, Mg²⁺, or Ca²⁺. Uncertainty bars represent SEM.

**Fig. 3.**
Structural bioinformatics predicts two classes of discrete actin filament-specific cation-binding sites. The actin filament on the left (PDB ID 3MFP “biological assembly”) is oriented with the barbed end at the bottom, and is colored by subunit. The central subunit is rendered as a cartoon showing the location of the predicted cation-binding sites. “Polymerization” sites (green spheres) have the highest prediction score from comparing WebFEATURE cation-binding site prediction results between the F-actin monomer (3MFP) and F-actin polymer (3MFP “biological assembly,” *Materials and Methods*). “Stiffness” sites (purple spheres) have the highest prediction score from comparing WebFEATURE cation-binding site prediction results between the G-actin monomer (PDB ID 1J6Z) and F-actin monomer (3MFP).

**Fig. 4.**
The “stiffness site” controls the cation dependence of actin filament rigidity. Bending persistence length (L_p) of A167E mutant yeast actin (Alexa 488 labeled) filaments increases with Mg²⁺-binding, whereas wt filaments shows no [Mg²⁺] dependence of L_p. Uncertainty bars represent SEM.

**Fig. 5.**
The “polymerization site” modulates the cation dependence of the critical concentration. T203C yeast actin shows little or no polymerization at 0.2 mM Mg²⁺ (C_c > 15 μM), C_c at 1 mM Mg²⁺ that is higher (C_c = 8.9 μM) than that of wt yeast actin, but a C_c value comparable to that of wt is achieved at 5 mM Mg²⁺. Uncertainty bars represent SEM.

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