Counter-Ion Binding To Actin

Eucaryotic cells contain a pool of cations that are bound to negatively charged metabolites, enzymes, and enzyme assemblies. Monovalent cations, such as sodium and potassium, form a cloud of counterions surrounding negatively charged biomolecules. The behavior of these ions can be described by continuum electrostatic (Poisson-Boltzmann) models of aqueous solutions near biomolecules. Divalent calcium and magnesium ions, however, are poised to bind to specific sites. Thereby, they neutralize any negatively charged surface at physiological ionic concentrations (Fig. 1). In such situations the implicit representation of ions near a protein surface by means of Poisson-Boltzmann theory is no longer valid, and explicit modeling of the ions is required. Unfortunately, low-affinity sites often remain undetected in crystallographic structures. At typical conditions of crystal buffers the sites are often occupied by monovalent ions that are difficult to distinguish from water molecules in the observed electron density map. Our goal is to develop a reliable method to predict low-affinity binding sites of calcium and magnesium ions. Once identified, divalent cations can be included in molecular dynamics, electrostatics, and Brownian dynamics calculations for an improved accuracy of the modeling.

Fig. 1 (click to enlarge): Actin monomers before and after counterion binding. Top: In the absence of calcium ions, the protein is negatively charged. Bottom: After binding of five counterions at predicted sites, the protein is neutralized. The isocontours (red) at -3 kcal/mol were calculated with the program Grasp at 0 ionic strength.

Our research is concerned, in particular, with the divalent-cation-driven polymerization of actin (described elsewhere). The aggregation requires the binding of divalent cations to five low-affinity sites (Kd ~15mM), neutralizing the monomers (charge at pH 7: -10 e). The location of these sites in the actin crystal structure is unknown. Brownian dynamics studies of actin-binding proteins and actin are under way in several research groups. As outlined above, these studies require the prediction of actin's divalent cation binding sites.

We have initiated searches for actin's five divalent cation binding sites and computed the free energy of binding at 10,000 equally-spaced test sites on actin's ion-accessible surface using the program UHBD (Fig. 2). These preliminary calculations will be followed by more extensive studies including side chain flexibility, ion cooperativity, and a ranking of the sites by the free energy of binding.

Fig. 2 (click to enlarge): Free energy of calcium binding to the actin monomer surface. Red: -5 kcal/mol; green: 0, blue; +5 kcal/mol.

Finally, we collaborate with the laboratory of Gerard Wong to determine directly the binding of counter-ion clouds to actin filament bundles by small-angle x-ray scattering (see reference below).


  1. Thomas E. Angelini, Hongjun Liang, Willy Wriggers and Gerard Wong. Like-charge attraction between polyelectrolytes induced by counterion charge density waves. PNAS, 2003, Vol. 100, pp. 8634-8637. [Abstract] [Article] [Software]

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