Effects of Binding Factors on Structural Elements in F-Actin

Abstract

Understanding the dynamics of the actin filament is essential to a detailed description of their interactions and role in the cell. Previous studies have linked the dynamic properties of actin filaments (F-actin) to three structural elements contributing to a hydrophobic pocket, namely, the hydrophobic loop, the DNase I binding loop, and the C-terminus. Here, we examine how these structural elements are influenced by factors that stabilize or destabilize F-actin, using site-directed spin-labeled (SDSL) electron paramagnetic resonance (EPR), fluorescence, and cross-linking techniques. Specifically, we employ cofilin, an actin destabilizing protein that binds and severs filaments, and phalloidin, a fungal toxin that binds and stabilizes F-actin. We find that cofilin shifts both the DNase I binding loop and the hydrophobic loop away from the C-terminus in F-actin, as demonstrated by weakened spin–spin interactions, and alters the environment of spin probes on residues of these two loops. In contrast, although phalloidin strongly stabilizes F-actin, it causes little or no local change in the environment of the loop residues. This indicates that the stabilizing effect of phalloidin is achieved mainly through constraining structural fluctuations in F-actin and suggests that factors and interactions that control these fluctuations have an important role in the cytoskeleton dynamics.

Actin filament remodeling by actin depolymerization factor/cofilin

“…The calculated average distance between residue Q41 of the DB loop and adjacent intrastrand subunit C-terminal residue C374 has been shown in a recent spectroscopic study to correlate with the effect of ADF/cofilin binding on actin filament dynamics (32). This experimental spectroscopic probe length is also directly calculable via simulation, and the present MD simulations yield similar results to those from experiment.…”

“…We note for clarity that this procedure was performed on each of the 11 or 13 actin subunits within each filament to obtain maximal sample size. The enhanced conformational flexibilities of the actin DB loop and C terminus are particularly interesting given their role identified in filament structure and dynamics (32).…”

“…A number of recent experimental studies of cofilactin have led to the development and support of the hypothesis that ADF/cofilin binds between subdomains 1 and 3 of actin, and that the origin of the increased flexibility of cofilactin (compared with bare filamentous actin) must have its roots in a modified interaction between subdomain 2 (SD2) and subdomain 1 (SD1) of the adjacent long-pitch helix subunit (10,13,15,32,34,35). We observed during simulation that ADF/cofilin binding does indeed result in reorganization of DB loop of subdomain 2 (SD2; Fig.…”

Metavinculin Tunes the Flexibility and the Architecture of Vinculin-Induced Bundles of Actin Filaments

“…3a-c) suggests that yeast actin mutants reliably report MVT induced changes in actin. Therefore, we focused on understanding the MVT induced changes in dynamic actin loops (H- (a.a. 262-274), D-(a.a. 40-50), W- (a.a. 167-170) and the C-terminus 32-35 . First, we asked whether MVT caused rearrangements in lateral and/or longitudinal interprotomer contacts.…”

“…Unlike MVT, ADF/cofilins significantly disrupt the interactions between actin interprotomer loops. Specifically interactions of actin’s subdomains 1 and 2 are disrupted by an increase in the plasticity of the D-loop upon cofilin binding 32,33,35,54,59-61 . This structural change accounts for the altered tilt and twist of cofilin decorated filaments observed by EM 60,61 , Cryo-EM 59 and AFM 40 .…”