Kien Xuan Ngo

Structural basis for actin assembly, activation of ATP hydrolysis, and delayed phosphate release

Assembled actin filaments support cellular signaling, intracellular trafficking, and cytokinesis. ATP hydrolysis triggered by actin assembly provides the structural cues for filament turnover in vivo. Here, we present the cryo-electron microscopic (cryo-EM) structure of filamentous actin (F-actin) in the presence of phosphate, with the visualization of some α-helical backbones and large side chains. A complete atomic model based on the EM map identified intermolecular interactions mediated by bound magnesium and phosphate ions. Comparison of the F-actin model with G-actin monomer crystal structures reveals a critical role for bending of the conserved proline-rich loop in triggering phosphate release following ATP hydrolysis. Crystal structures of G-actin show that mutations in this loop trap the catalytic site in two intermediate states of the ATPase cycle. The combined structural information allows us to propose a detailed molecular mechanism for the biochemical events, including actin polymerization and ATPase activation, critical for actin filament dynamics.

Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy

High-speed atomic force microscopy was employed to observe structural changes in actin filaments induced by cofilin binding. Consistent with previous electron and fluorescence microscopic studies, cofilin formed clusters along actin filaments, where the filaments were 2-nm thicker and the helical pitch was ∼25% shorter, compared to control filaments. Interestingly, the shortened helical pitch was propagated to the neighboring bare zone on the pointed-end side of the cluster, while the pitch on the barbed-end side was similar to the control. Thus, cofilin clusters induce distinctively asymmetric conformational changes in filaments. Consistent with the idea that cofilin favors actin structures with a shorter helical pitch, cofilin clusters grew unidirectionally toward the pointed-end of the filament. Severing was often observed near the boundaries between bare zones and clusters, but not necessarily at the boundaries. DOI: http://dx.doi.org/10.7554/eLife.04806.001

Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin.

Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

Uni-directional Propagation of Structural Changes in Actin Filaments

When a protein molecule is bound with another, its structure is likely to change in one way or the other. The structure of a protein molecule in a protein complex is also likely to change when binding partner in the complex undergoes a conformational change. It is therefore no surprise that binding of an actin-binding protein to a protomer in an actin filament changes the structure of that actin protomer, and that the resultant conformational change in the actin protomer affects the structure of the neighboring protomers in the same filament. Moreover, eukaryotic actin appears to have evolved to efficiently spread the conformational change in the actin protomer initially bound with actin-binding protein over a long distance along the filament (cooperative conformational change), as has been observed in the cases of cofilin- and myosin-induced cooperative conformational changes. We speculate that the high degree of cooperativity in conformational changes in actin filaments enables cooperative binding of actin-binding proteins, which is necessary for actin filaments to perform specific functions by selectively interacting with a subset of actin-binding proteins among the large number of actin-binding proteins present in the cell. Interestingly, cooperative conformational changes propagate to only one direction along the filament, at least in the cases of cofilin and myosin II-induced conformational changes. Functional significance of those uni-directional conformational changes in actin filaments is not known, but we propose that they play roles in directional signal transmission along one-dimensional polymer in cells, or in force generation by myosin.