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Cations Stiffen Actin Filaments by Adhering a Key Structural Element to Adjacent Subunits - PubMed

  • ️Fri Jan 01 2016

Cations Stiffen Actin Filaments by Adhering a Key Structural Element to Adjacent Subunits

Glen M Hocky et al. J Phys Chem B. 2016.

Abstract

Ions regulate the assembly and mechanical properties of actin filaments. Recent work using structural bioinformatics and site-specific mutagenesis favors the existence of two discrete and specific divalent cation binding sites on actin filaments, positioned in the long axis between actin subunits. Cation binding at one site drives polymerization, while the other modulates filament stiffness and plays a role in filament severing by the regulatory protein, cofilin. Existing structural methods have not been able to resolve filament-associated cations, and so in this work we turn to molecular dynamics simulations to suggest a candidate binding pocket geometry for each site and to elucidate the mechanism by which occupancy of the "stiffness site" affects filament mechanical properties. Incorporating a magnesium ion in the "polymerization site" does not seem to require any large-scale change to an actin subunit's conformation. Binding of a magnesium ion in the "stiffness site" adheres the actin DNase-binding loop (D-loop) to its long-axis neighbor, which increases the filament torsional stiffness and bending persistence length. Our analysis shows that bound D-loops occupy a smaller region of accessible conformational space. Cation occupancy buries key conserved residues of the D-loop, restricting accessibility to regulatory proteins and enzymes that target these amino acids.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1

Polymerization (yellow) and stiffness (purple) cations are added in putative binding sites between actin subunits in the positions shown. Beads labeled 1–4 show the center of mass positions of the four actin subdomains as defined in

Table S3

, while bead 5 shows the center of mass of the D-loop (residues 40–51). A subscript indicates the relative subunit position in the filament (increasing toward the “pointed” end). Also shown is the φ2134 dihedral angle, which is used to measure the planarity of an actin monomer.

Figure 2
Figure 2

Typical snapshots from simulations of the Namba model with (A,C) and without (B,D) additional coordinated Mg2+ ions. Panels A and B show an overview, and C and D show a closer view. (A,C) Polymerization cations shown in yellow, and stiffness cations are shown in purple. Protein residues and water molecules within 5 Å of the central cations are atomically resolved, as are residues D292′ and K61, which form a salt bridge when the stiffness ion is added. (B,D) Residue positions that will form the core of the polymerization site (E205, D286′, D286′, and I287′) and form the stiffness site (Q49, K50, D51, E57, E167′) are shown in detail, as well as water within 5 Å of D286′, D288′ and E167′. The side chain of K61 is oriented such that it forms a salt bridge with E167′.

Figure 3
Figure 3

(A) Cation binding geometry is six coordinate and has high symmetry. Vertical dashed line shows the value of Q4 for an ideal octahedron. The polymerization site (P), while still symmetric, is more distorted than the binding in the stiffness site (S). (B) Distance between charged atoms in residues suspected to form a salt bridge in actin. Top–salt bridge forms after removal of stiffness site Mg2+ ion. Bottom–alternative salt bridge is persistent in the cation-coordinated system, and breaks when the stiffness ions are removed.

Figure 4
Figure 4

Coarse-grained distribution functions. Coarse-grained subunit definitions are illustrated in Figure 1. (A) Twist angle between adjacent subunits in the filament. (B) Average longitudinal distance between adjacent actin subunits in the filament. (C) Flattening dihedral angle in the actin subunits. (D) Angle formed between subdomains 2 and 4 in an actin filament, and subdomain 3 in the actin two subunits away. (E) Distance between the actin D-loop and the coordinated subdomain 3. (F) Distance within a subunit between subdomain 3 and the center-of-mass of the D-loop.

Figure 5
Figure 5

D-loop fluctuations. (A) First two principal components from dPCA analysis reveal three states, folded, unfolded and cofilin-like. (B) DM analysis confirms existence of same three states. Results for filament with coordinated Mg2+ are restricted to a subset of unfolded structures. (C) Typical snapshots taken from the different states identified in (A). All are oriented from N-term to C-term going from left to right. Unfolded conformations have PC1 > −1 and PC2 < 2, folded conformations have PC > 2.5, and cofilin-like structures have PC1 < −1. (D,E) Interface between segments with and without coordinated stiffness ion. Twenty snapshots for D-loop and adjacent residues (40–62) are shown every 2.5 ps from a representative molecular dynamics trajectory. The D-loop conformations in the ion-bound system are more tightly bound to the adjacent subdomain 3 (Figure 4).

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