Taro Q.P. Noguchi

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.

A Novel System for Expressing Toxic Actin Mutants in Dictyostelium and Purification and Characterization of a Dominant Lethal Yeast Actin Mutant

We have developed a novel system for expressing recombinant actin in Dictyostelium. In this system, the C terminus of actin is fused to thymosin β via a glycine-based linker. The fusion protein is purified using a His tag attached to the thymosin β moiety and then cleaved by chymotrypsin immediately after the native final residue of actin to yield intact actin. Wild-type actin prepared in this way was functionally normal in terms of its polymerization kinetics and muscle myosin-mediated motility. We expected that this system would be particularly useful for expressing toxic actin mutants, because the actin moiety of the fusion protein is unlikely to interact with the actin cytoskeleton of the host cells. We therefore chose to express the E206A/R207A/E208A mutant, which appears to be dominant lethal in yeast, as a model case of a toxic actin mutant that is difficult to express. We found that the E206A/R207A/E208A mutant could be expressed and purified with a yield comparable to the wild-type molecule (3–4 mg/20 g cells), even though green fluorescent protein-fused actin carrying the E206A/R207A/E208A mutation was expressed at a much lower level than wild-type actin. Purified E206A/R207A/E208A actin did not polymerize, even in the presence of muscle actin; however, it accelerated polymerization of muscle actin and inhibited the nucleating and severing activities of gelsolin. Given that the location of the substituted residues is near the pointed end face of the mutant, we suggest that E206A/R207A/E208A actin behaves like a weak pointed end-capping protein that perturbs the actin cytoskeleton of the host cells.

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.

Rapid Nucleotide Exchange Renders Asp-11 Mutant Actins Resistant to Depolymerizing Activity of Cofilin, Leading to Dominant Toxicity in Vivo

Background: Mutation of Asp-11 is dominant negative in yeast and human actins. Results: Mutant actins exchange bound nucleotides rapidly, cannot bind cofilin, and cofilin-induced depolymerization of mutant and wild type copolymers is slow. Conclusion: Rapid nucleotide exchange with exogenous ATP inhibits cofilin-mediated depolymerization of copolymers, leading to dominant toxicity. Significance: Mechanism of a dominant negative actin mutation is elucidated. Conserved Asp-11 of actin is a part of the nucleotide binding pocket, and its mutation to Gln is dominant lethal in yeast, whereas the mutation to Asn in human α-actin dominantly causes congenital myopathy. To elucidate the molecular mechanism of those dominant negative effects, we prepared Dictyostelium versions of D11N and D11Q mutant actins and characterized them in vitro. D11N and D11Q actins underwent salt-dependent reversible polymerization, although the resultant polymerization products contained small anomalous structures in addition to filaments of normal appearance. Both monomeric and polymeric D11Q actin released bound nucleotides more rapidly than the wild type, and intriguingly, both monomeric and polymeric D11Q actins hardly bound cofilin. The deficiency in cofilin binding can be explained by rapid exchange of bound nucleotide with ATP in solution, because cofilin does not bind ATP-bound actin. Copolymers of D11Q and wild type actins bound cofilin, but cofilin-induced depolymerization of the copolymers was slower than that of wild type filaments, which may presumably be the primary reason why this mutant actin is dominantly toxic in vivo. Purified D11N actin was unstable, which made its quantitative biochemical characterization difficult. However, monomeric D11N actin released nucleotides even faster than D11Q, and we speculate that D11N actin also exerts its toxic effects in vivo through a defective interaction with cofilin. We have recently found that two other dominant negative actin mutants are also defective in cofilin binding, and we propose that the defective cofilin binder is a major class of dominant negative actin mutants.

Screening of novel dominant negative mutant actins using glycine targeted scanning identifies G146V actin that cooperatively inhibits cofilin binding

A number of studies suggested that the structure of actin filaments changes by interaction with actin-binding proteins such as cofilin and myosin, and that the conformational changes of the actin subunits within a filament are cooperative. To understand the functions of these cooperative conformational changes induced by actin-binding proteins, we sought to obtain dominant negative mutant actins impaired in cooperative conformational changes. A series of mutant actin genes in which glycine residues in actin were systematically substituted by valine residues were constructed, and were expressed individually in yeast cells that carry a wild-type endogenous actin gene. Six dominant negative actin mutations were identified on the basis of growth inhibition. Among them, G146V mutation was chosen for further biochemical analysis because the Gly146 residue is located at the strategic hinge position connecting the large and small domains of an actin molecule. We found that G146V actin filaments hardly bind cofilin, consistent with a previous suggestion that cofilin binding causes conformational changes of actin around Gly146 (Galkin et al. [3]). Notably, copolymer that consists of 1:10 mixture of the mutant and wild-type actin molecules showed significantly reduced affinity for cofilin, suggesting that G146V mutant actin affects the conformation of neighboring wild-type actin within a filament, and inhibits cofilin binding.

G146V mutation at the hinge region of actin reveals a myosin class-specific requirement of actin conformations for motility

Background: The roles of conformational changes of actin in myosin motility are unclear. Results: A G146V mutation in actin, which perturbed its conformation, impaired force generation by myosin II, but not by myosin V. Conclusion: Conformational changes of actin involving Gly-146 have critical roles in motility of myosin II, but not of myosin V. Significance: The mechanism of motility may be different between myosin types. The G146V mutation in actin is dominant lethal in yeast. G146V actin filaments bind cofilin only minimally, presumably because cofilin binding requires the large and small actin domains to twist with respect to one another around the hinge region containing Gly-146, and the mutation inhibits that twisting motion. A number of studies have suggested that force generation by myosin also requires actin filaments to undergo conformational changes. This prompted us to examine the effects of the G146V mutation on myosin motility. When compared with wild-type actin filaments, G146V filaments showed a 78% slower gliding velocity and a 70% smaller stall force on surfaces coated with skeletal heavy meromyosin. In contrast, the G146V mutation had no effect on either gliding velocity or stall force on myosin V surfaces. Kinetic analyses of actin-myosin binding and ATPase activity indicated that the weaker affinity of actin filaments for myosin heads carrying ADP, as well as reduced actin-activated ATPase activity, are the cause of the diminished motility seen with skeletal myosin. Interestingly, the G146V mutation disrupted cooperative binding of myosin II heads to actin filaments. These data suggest that myosin-induced conformational changes in the actin filaments, presumably around the hinge region, are involved in mediating the motility of skeletal myosin but not myosin V and that the specific structural requirements for the actin subunits, and thus the mechanism of motility, differ among myosin classes.

Dominant negative mutant actins identified in flightless Drosophila can be classified into three classes.

Strongly dominant negative mutant actins, identified by An and Mogami (An, H. S., and Mogami, K. (1996) J. Mol. Biol. 260, 492–505), in the indirect flight muscle of Drosophila impaired its flight, even when three copies of the wild-type gene were present. Understanding how these strongly dominant negative mutant actins disrupt the function of wild-type actin would provide useful information about the molecular mechanism by which actin functions in vivo. Here, we expressed and purified six of these strongly dominant negative mutant actins in Dictyostelium and classified them into three groups based on their biochemical phenotypes. The first group, G156D, G156S, and G268D actins, showed impaired polymerization and a tendency to aggregate under conditions favoring polymerization. G63D actin of the second group was also unable to polymerize but, unlike those in the first group, remained soluble under polymerizing conditions. Kinetic analyses using G63D actin or G63D actin·gelsolin complexes suggested that the pointed end surface is defective, which would alter the polymerization kinetics of wild-type actin when mixed and could affect formation of thin filament structures in indirect flight muscle. The third group, R95C and E226K actins, was normal in terms of polymerization, but their motility on heavy meromyosin surfaces in the presence of tropomyosin-troponin indicated altered sensitivity to Ca2+. Cofilaments in which R95C or E226K actins were copolymerized with a 3-fold excess of wild-type actin also showed altered Ca2+ sensitivity in the presence of tropomyosin-troponin.

The Role of Structural Dynamics of Actin in Class-Specific Myosin Motility

The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin.

ADF/Cofilin Is Not Essential but Is Critically Important for Actin Activities during Phagocytosis in Tetrahymena thermophila

ABSTRACT ADF/cofilin is a highly conserved actin-modulating protein. Reorganization of the actin cytoskeleton in vivo through severing and depolymerizing of F-actin by this protein is essential for various cellular events, such as endocytosis, phagocytosis, cytokinesis, and cell migration. We show that in the ciliate Tetrahymena thermophila, the ADF/cofilin homologue Adf73p associates with actin on nascent food vacuoles. Overexpression of Adf73p disrupted the proper localization of actin and inhibited the formation of food vacuoles. In vitro, recombinant Adf73p promoted the depolymerization of filaments made of T. thermophila actin (Act1p). Knockout cells lacking the ADF73 gene are viable but grow extremely slowly and have a severely decreased rate of food vacuole formation. Knockout cells have abnormal aggregates of actin in the cytoplasm. Surprisingly, unlike the case in animals and yeasts, in Tetrahymena, ADF/cofilin is not required for cytokinesis. Thus, the Tetrahymena model shows promise for future studies of the role of ADF/cofilin in vivo.