Structural evidence for the roles of divalent cations in actin polymerization and activation of ATP hydrolysis

Figures

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  Fig. 1.. Crystal structure of the actin/CoblNW complex at a 2.0-Å resolution. (A) Actin, light blue cartoon/surface. CoblNW, α-carbons purple trace. Ca2+, green spheres. ATP is shown as orange sticks. (B) Closeup of the D288/D286 Ca2+ site. Residues from the two symmetry-related molecules are shown in light blue and blue, associated with electron density, gray and blue mesh, respectively, contoured at 1 σ. Water molecules are shown as red spheres. (C) The ATP-associated Ca2+ site. (D) Waters close to the ATP γ-phosphate.

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  Fig. 2.. Review of divalent cation binding sites found in the PDB crystal structure library. F-actin is represented in the Middle (PDB 3J8A), in which four actin protomers are illustrated as surfaces (blue, green, orange, and pink), and the central protomer as a light blue cartoon. (A–E “crystal structures”) Enlargements of the cation binding sites from crystal structures are shown as green or yellow sticks, and Mg2+ and Ca2+ as black and green spheres, respectively. Sites that were previously described are boxed by red squares (A, C, and E) and those that were additionally observed in several crystal structures are boxed by green squares (B and D). (A–E “MD”) Mg2+ coordination after 100-ns MD simulation, in respective conditions. A and C are the upper and lower polymerization site, and E is the lower stiffness site (the upper site is identical and omitted for clarity).

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  Fig. 3.. Symmetry conservation around the ATP-bound Mg2+ in MD. (A and C) Initial MD models of ATP and ATP-bound Mg2+ for F- and G-actin, respectively. (B and D) Final snapshots after a 100-ns long MD simulation of F- and G-actin, respectively. ATP, sticks. Mg2+, black spheres. Water, red spheres. (E and F) Analysis of different angles (brown lines) and distances (blue and green lines) around ATP-bound Mg2+, over time, for F- and G-actin, respectively. Simulations were performed in 120 mM KCl and 1 mM MgCl2.

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  Fig. 4.. MD analysis of H2O close to the γ-phosphate and Gln137. (A, C, and E) Schematic representation of analysis conditions applied around ATP and Gln137. (B, D, and F) Cumulative probability of H2O with applied conditions from A, C, and E. (G) G-actin model, resulting from the combination of actin-DNaseI structure (PDB 1ATN) and ATP and ATP-bound Mg2+ of PDB 2V52. (H) Typical snapshot of MD simulation frame, satisfying all conditions from E. ATP and actin, sticks. Waters coordinated by Mg2+ are shown as blue, green, and purple spheres.