Structures and mechanisms of actin ATP hydrolysis.

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) PG-O3B bond cleavage; 3) four concomitant events: W1-PO3- formation, OH- and proton cleavage, nucleophilic attack by the OH- against PG, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi-bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Novel inter-domain Ca2+-binding site in the gelsolin superfamily protein fragmin.

Gelsolin superfamily proteins, consisting of multiple domains (usually six), sever actin filaments and cap the barbed ends in a Ca2+-dependent manner. Two types of evolutionally conserved Ca2+-binding sites have been identified in this family; type-1 (between gelsolin and actin) and type-2 (within the gelsolin domain). Fragmin, a member in the slime mold Physarum polycephalum, consists of three domains (F1-F3) that are highly similar to the N-terminal half of mammalian gelsolin (G1-G3). Despite their similarities, the two proteins exhibit a significant difference in the Ca2+ dependency; F1-F3 absolutely requires Ca2+ for the filament severing whereas G1-G3 does not. In this study, we examined the strong dependency of fragmin on Ca2+ using biochemical and structural approaches. Our co-sedimentation assay demonstrated that Ca2+ significantly enhanced the binding of F2-F3 to actin. We determined the crystal structure of F2-F3 in the presence of Ca2+. F2-F3 binds a total of three calcium ions; while two are located in type-2 sites within F2 or F3, the remaining one resides between the F2 long helix and the F3 short helix. The inter-domain Ca2+-coordination appears to stabilize F2-F3 in a closely packed configuration. Notably, the F3 long helix exhibits a bent conformation which is different from the straight G3 long helix in the presence of Ca2+. Our results provide the first structural evidence for the existence of an unconventional Ca2+-binding site in the gelsolin superfamily proteins.

Novel actin filaments from Bacillus thuringiensis form nanotubules for plasmid DNA segregation.

Here we report the discovery of a bacterial DNA-segregating actin-like protein (BtParM) from Bacillus thuringiensis, which forms novel antiparallel, two-stranded, supercoiled, nonpolar helical filaments, as determined by electron microscopy. The BtParM filament features of supercoiling and forming antiparallel double-strands are unique within the actin fold superfamily, and entirely different to the straight, double-stranded, polar helical filaments of all other known ParMs and of eukaryotic F-actin. The BtParM polymers show dynamic assembly and subsequent disassembly in the presence of ATP. BtParR, the DNA-BtParM linking protein, stimulated ATP hydrolysis/phosphate release by BtParM and paired two supercoiled BtParM filaments to form a cylinder, comprised of four strands with inner and outer diameters of 57 Å and 145 Å, respectively. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable features to the eukaryotic chromosome-segregating microtubule.

Structural basis for the slow dynamics of the actin filament pointed end.

The actin filament has clear polarity where one end, the pointed end, has a much slower polymerization and depolymerization rate than the other end, the barbed end. This intrinsic difference of the ends significantly affects all actin dynamics in the cell, which has central roles in a wide spectrum of cellular functions. The detailed mechanism underlying this difference has remained elusive, because high-resolution structures of the filament ends have not been available. Here, we present the structure of the actin filament pointed end obtained using a single particle analysis of cryo-electron micrographs. We determined that the terminal pointed end subunit is tilted towards the penultimate subunit, allowing specific and extra loop-to-loop inter-strand contacts between the two end subunits, which is not possible in other parts of the filament. These specific contacts prevent the end subunit from dissociating. For elongation, the loop-to-loop contacts also inhibit the incorporation of another actin monomer at the pointed end. These observations are likely to account for the less dynamic pointed end.

Internal dynamics of F-actin and myosin subfragment-1 studied by quasielastic neutron scattering.

Various biological functions related to cell motility are driven by the interaction between the partner proteins, actin and myosin. To obtain insights into how this interaction occurs, the internal dynamics of F-actin and myosin subfragment-1 (S1) were characterized by the quasielastic neutron scattering measurements on the solution samples of F-actin and S1. Contributions of the internal motions of the proteins to the scattering spectra were separated from those of the global macromolecular diffusion. Analysis of the spectra arising from the internal dynamics showed that the correlation times of the atomic motions were about two times shorter for F-actin than for S1, suggesting that F-actin fluctuates more rapidly than S1. It was also shown that the fraction of the immobile atoms is larger for S1 than for F-actin. These results suggest that F-actin actively facilitates the binding of myosin by utilizing the more frequent conformational fluctuations than those of S1.

The nature of the globular- to fibrous-actin transition.

Actin plays crucial parts in cell motility through a dynamic process driven by polymerization and depolymerization, that is, the globular (G) to fibrous (F) actin transition. Although our knowledge about the actin-based cellular functions and the molecules that regulate the G- to F-actin transition is growing, the structural aspects of the transition remain enigmatic. We created a model of F-actin using X-ray fibre diffraction intensities obtained from well oriented sols of rabbit skeletal muscle F-actin to 3.3 A in the radial direction and 5.6 A along the equator. Here we show that the G- to F-actin conformational transition is a simple relative rotation of the two major domains by about 20 degrees. As a result of the domain rotation, the actin molecule in the filament is flat. The flat form is essential for the formation of stable, helical F-actin. Our F-actin structure model provides the basis for understanding actin polymerization as well as its molecular interactions with actin-binding proteins.

Role of the actin Ala-108-Pro-112 loop in actin polymerization and ATPase activities.

Actin plays fundamental roles in a variety of cell functions in eukaryotic cells. The polymerization-depolymerization cycle, between monomeric G-actin and fibrous F-actin, drives essential cell processes. Recently, we proposed the atomic model for the F-actin structure and found that actin was in the twisted form in the monomer and in the untwisted form in the filament. To understand how the polymerization process is regulated (Caspar, D. L. (1991) Curr. Biol. 1, 30-32), we need to know further details about the transition from the twisted to the untwisted form. For this purpose, we focused our attention on the Ala-108-Pro-112 loop, which must play crucial roles in the transition, and analyzed the consequences of the amino acid replacements on the polymerization process. As compared with the wild type, the polymerization of P109A was accelerated in both the nucleation and the elongation steps, and this was attributed to an increase in the frequency factor of the Arrhenius equation. The multiple conformations allowed by the substitution presumably resulted in the effective formation of the collision complex, thus accelerating polymerization. On the other hand, the A108G mutation reduced the rates of both nucleation and elongation due to an increase in the activation energy. In the cases of polymerization acceleration and deceleration, each functional aberration is attributed to a distinct elementary process. The rigidity of the loop, which mediates neither too strong nor too weak interactions between subdomains 1 and 3, might play crucial roles in actin polymerization.

Cooperative and non-cooperative conformational changes of F-actin induced by cofilin.

Cofilin is an actin-binding protein that promotes F-actin depolymerization. It is well-known that cofilin-coated F-actin is more twisted than naked F-actin, and that the protomer is more tilted. However, the means by which the local changes induced by the binding of individual cofilin proteins proceed to the global conformational changes of the whole F-actin molecule remain unknown. Here we investigated the cofilin-induced changes in several parts of F-actin, through site-directed spin-label electron paramagnetic resonance spectroscopy analyses of recombinant actins containing single reactive cysteines. We found that the global, cooperative conformational changes induced by cofilin-binding, which were detected by the spin-label attached to the Cys374 residue, occurred without the detachment of the D-loop in subdomain 2 from the neighboring protomer. The two processes of local and global changes do not necessarily proceed in sequence.

Coupling of the hydration water dynamics and the internal dynamics of actin detected by quasielastic neutron scattering.

In order to characterize dynamics of water molecules around F-actin and G-actin, quasielastic neutron scattering experiments were performed on powder samples of F-actin and G-actin, hydrated either with D(2)O or H(2)O, at hydration ratios of 0.4 and 1.0. By combined analysis of the quasielastic neutron scattering spectra, the parameter values characterizing the dynamics of the water molecules in the first hydration layer and those of the water molecules outside of the first layer were obtained. The translational diffusion coefficients (D(T)) of the hydration water in the first layer were found to be 1.2×10(-5) cm(2)/s and 1.7×10(-5) cm(2)/s for F-actin and G-actin, respectively, while that for bulk water was 2.8×10(-5) cm(2)/s. The residence times were 6.6 ps and 5.0 ps for F-actin and G-actin, respectively, while that for bulk water was 0.62 ps. These differences between F-actin and G-actin, indicating that the hydration water around G-actin is more mobile than that around F-actin, are in concert with the results of the internal dynamics of F-actin and G-actin, showing that G-actin fluctuates more rapidly than F-actin. This implies that the dynamics of the hydration water is coupled to the internal dynamics of the actin molecules. The D(T) values of the water molecules outside of the first hydration layer were found to be similar to that of bulk water though the residence times are strongly affected by the first hydration layer. This supports the recent observation on intracellular water that shows bulk-like behavior.

Mutagenic analysis of actin reveals the mechanism of His161 flipping that triggers ATP hydrolysis.

The dynamic assembly of actin is controlled by the hydrolysis of ATP, bound to the center of the molecule. Upon polymerization, actin undergoes a conformational change from the monomeric G-form to the fibrous F-form, which is associated with the flipping of the side chain of His161 toward ATP. His161 flipping from the gauche-minus to gauche-plus conformation leads to a rearrangement of the active site water molecules, including ATP attacking water (W1), into an orientation capable of hydrolysis. We previously showed that by using a human cardiac muscle α-actin expression system, mutations in the Pro-rich loop residues (A108G and P109A) and in a residue that was hydrogen-bonded to W1 (Q137A) affect the rate of polymerization and ATP hydrolysis. Here, we report the crystal structures of the three mutant actins bound to AMPPNP or ADP-Pi determined at a resolution of 1.35-1.55 Å, which are stabilized in the F-form conformation with the aid of the fragmin F1 domain. In A108G, His161 remained non-flipped despite the global actin conformation adopting the F-form, demonstrating that the side chain of His161 is flipped to avoid a steric clash with the methyl group of A108. Because of the non-flipped His161, W1 was located away from ATP, similar to G-actin, which was accompanied by incomplete hydrolysis. In P109A, the absence of the bulky proline ring allowed His161 to be positioned near the Pro-rich loop, with a minor influence on ATPase activity. In Q137A, two water molecules replaced the side-chain oxygen and nitrogen of Gln137 almost exactly at their positions; consequently, the active site structure, including the W1 position, is essentially conserved. This seemingly contradictory observation to the reported low ATPase activity of the Q137A filament could be attributed to a high fluctuation of the active site water. Together, our results suggest that the elaborate structural design of the active site residues ensures the precise control of the ATPase activity of actin.

Molecular structure of the ParM polymer and the mechanism leading to its nucleotide-driven dynamic instability.

ParM is a prokaryotic actin homologue, which ensures even plasmid segregation before bacterial cell division. In vivo, ParM forms a labile filament bundle that is reminiscent of the more complex spindle formed by microtubules partitioning chromosomes in eukaryotic cells. However, little is known about the underlying structural mechanism of DNA segregation by ParM filaments and the accompanying dynamic instability. Our biochemical, TIRF microscopy and high-pressure SAX observations indicate that polymerization and disintegration of ParM filaments is driven by GTP rather than ATP and that ParM acts as a GTP-driven molecular switch similar to a G protein. Image analysis of electron micrographs reveals that the ParM filament is a left-handed helix, opposed to the right-handed actin polymer. Nevertheless, the intersubunit contacts are similar to those of actin. Our atomic model of the ParM-GMPPNP filament, which also fits well to X-ray fibre diffraction patterns from oriented gels, can explain why after nucleotide release, large conformational changes of the protomer lead to a breakage of intra- and interstrand interactions, and thus to the observed disintegration of the ParM filament after DNA segregation.

Distinct conformations of in vitro and in vivo amyloids of huntingtin-exon1 show different cytotoxicity.

A hallmark of polyglutamine diseases, including Huntington disease (HD), is the formation of beta-sheet-rich aggregates, called amyloid, of causative proteins with expanded polyglutamines. However, it has remained unclear whether the polyglutamine amyloid is a direct cause or simply a secondary manifestation of the pathology. Here we show that huntingtin-exon1 (thtt) with expanded polyglutamines remarkably misfolds into distinct amyloid conformations under different temperatures, such as 4 degrees C and 37 degrees C. The 4 degrees C amyloid has loop/turn structures together with mostly beta-sheets, including exposed polyglutamines, whereas the 37 degrees C amyloid has more extended and buried beta-sheets. By developing a method to efficiently introduce amyloid into mammalian cells, we found that the formation of the 4 degrees C amyloid led to substantial toxicity, whereas the toxic effects of the 37 degrees C amyloid were very small. Importantly, thtt amyloids in different brain regions of HD mice also had distinct conformations. The thermolabile thtt amyloid with loop/turn structures in the striatum showed higher toxicity, whereas the rigid thtt amyloid with more extended beta-sheets in the hippocampus and cerebellum had only mild toxic effects. These studies show that the thtt protein with expanded polyglutamines can misfold into distinct amyloid conformations and, depending on the conformations, the amyloids can be either toxic or nontoxic. Thus, the amyloid conformation of thtt may be a critical determinant of cytotoxicity in HD.

Internal motions of actin characterized by quasielastic neutron scattering.

Quasielastic neutron scattering (QENS) experiments were carried out on powders of F-actin and G-actin hydrated with D(2)O to characterize the internal dynamics on the picosecond time scale and the Ångstrom length scale. To investigate the effects of hydration, the measurements were done on samples at hydration ratio (h) of 0.4 (mg D(2)O/mg protein), containing only the first layer of hydration water, and at h = 1.0, containing more layers of water. The QENS spectra, obtained from the measurements at two energy resolutions of 110 and 15 µeV, indicated that the internal motions of both F-actin and G-actin have distributions of motions with distinct correlation times and amplitudes. Increasing hydration changes relative populations of these distinct motions. The effects of hydration were shown to be different between F-actin and G-actin. Elastic incoherent neutron scattering measurements provided the concerted results. The observed effects were interpreted in terms of the dynamical heterogeneity of the actin molecule: in G-actin, more surface loops become flexible and undergo diffusive motions of large amplitudes, whereas in F-actin the molecular interactions that keep the polymerized state suppress the large motions of the surface loops involved with polymerization so that the population of atoms undergoing large motions can increase only to a lesser degree.

Reconstitution of the muscle thin filament from recombinant troponin components and the native thin filaments.

We have developed a technique by which muscle thin filaments are reconstituted from the recombinant troponin components and the native thin filaments. By this technique, the reconstituted troponin complex is exchanged into the native thin filaments in the presence of 20% glycerol and 0.3M KCl at pH 6.2. More than 90% of endogenous troponin complex was replaced with the recombinant troponin complex. Structural integrity and Ca(2+) sensitivity of the reconstituted thin filament prepared by this technique was confirmed by X-ray fiber diffraction measurements and the thin filament-activated myosin subfragment 1 ATPase measurements, respectively.

D-Loop Mutation G42A/G46A Decreases Actin Dynamics.

Depolymerization and polymerization of the actin filament are indispensable in eukaryotes. The DNase I binding loop (D-loop), which forms part of the interface between the subunits in the actin filament, is an intrinsically disordered loop with a large degree of conformational freedom. Introduction of the double mutation G42A/G46A to the D-loop of the beta cytoskeletal mammalian actin restricted D-loop conformational freedom, whereas changes to the critical concentration were not large, and no major structural changes were observed. Polymerization and depolymerization rates at both ends of the filament were reduced, and cofilin binding was inhibited by the double mutation. These results indicate that the two glycines at the tip of the D-loop are important for actin dynamics, most likely by contributing to the large degree of conformational freedom.

Nucleotide-dependence of G-actin conformation from multiple molecular dynamics simulations and observation of a putatively polymerization-competent superclosed state.

The assembly of monomeric G-actin into filamentous F-actin is nucleotide dependent: ATP-G-actin is favored for filament growth at the "barbed end" of F-actin, whereas ADP-G-actin tends to dissociate from the "pointed end." Structural differences between ATP- and ADP-G-actin are examined here using multiple molecular dynamics simulations. The "open" and "closed" conformational states of G-actin in aqueous solution are characterized, with either ATP or ADP in the nucleotide binding pocket. With both ATP and ADP bound, the open state closes in the absence of actin-bound profilin. The position of the nucleotide in the protein is found to be correlated with the degree of opening of the active site cleft. Further, the simulations reveal the existence of a structurally well-defined, compact, "superclosed" state of ATP-G-actin, as yet unseen crystallographically and absent in the ADP-G-actin simulations. The superclosed state resembles structurally the actin monomer in filament models derived from fiber diffraction and is putatively the polymerization competent conformation of ATP-G-actin.

Structural Polymorphism of Actin.

Information on the structural polymorphism of a protein is essential to understand the mechanisms of how it functions at an atomic level. Numerous studies on actin have accumulated substantial amounts of information about its polymorphism, and there are over 200 published atomic structures of different forms of actin using crystallography, fiber diffraction, and electron microscopy. To characterize all the reported structures, we proposed simple parameters based on the discrete rigid bodies within the actin molecule and identified four conformation groups by cluster analysis: the F-form in naked F-actin, the C-form in cofilactin, the O-form in profilin-actin, and the G-form in the majority of actin-containing crystal structures. The G-form group included the most variations, but each conformational variation was convertible via a thermal fluctuation, whereas the F- and C-forms were not accessible from the G-form. The convertibility and accessibility of the structures were evaluated using molecular dynamics simulations. Information about conformational conversion among each group is useful for understanding the mechanisms of actin function.

Differences in internal dynamics of actin under different structural states detected by neutron scattering.

F-actin, a helical polymer formed by polymerization of the monomers (G-actin), plays crucial roles in various aspects of cell motility. Flexibility of F-actin has been suggested to be important for such a variety of functions. Understanding the flexibility of F-actin requires characterization of a hierarchy of dynamical properties, from internal dynamics of the actin monomers through domain motions within the monomers and relative motions between the monomers within F-actin to large-scale motions of F-actin as a whole. As a first step toward this ultimate purpose, we carried out elastic incoherent neutron scattering experiments on powders of F-actin and G-actin hydrated with D(2)O and characterized the internal dynamics of F-actin and G-actin. Well established techniques and analysis enabled the extraction of mean-square displacements and their temperature dependence in F-actin and in G-actin. An effective force constant analysis with a model consisting of three energy states showed that two dynamical transitions occur at approximately 150 K and approximately 245 K, the former of which corresponds to the onset of anharmonic motions and the latter of which couples with the transition of hydration water. It is shown that behavior of the mean-square displacements is different between G-actin and F-actin, such that G-actin is "softer" than F-actin. The differences in the internal dynamics are detected for the first time between the different structural states (the monomeric state and the polymerized state). The different behavior observed is ascribed to the differences in dynamical heterogeneity between F-actin and G-actin. Based on structural data, the assignment of the differences observed in the two samples to dynamics of specific loop regions involved in the polymerization of G-actin into F-actin is proposed.

Polymerization and depolymerization of actin with nucleotide states at filament ends.

Polymerization induces hydrolysis of ATP bound to actin, followed by γ-phosphate release, which helps advance the disassembly of actin filaments into ADP-G-actin. Mechanical understanding of this correlation between actin assembly and ATP hydrolysis has been an object of intensive studies in biochemistry and structural biology for many decades. Although actin polymerization and depolymerization occur only at either the barbed or pointed ends and the kinetic and equilibrium properties are substantially different from each other, characterizing their properties is difficult to do by bulk assays, as these assays report the average of all actin filaments in solution and are therefore not able to discern the properties of individual actin filaments. Biochemical studies of actin polymerization and hydrolysis were hampered by these inherent properties of actin filaments. Total internal reflection fluorescence (TIRF) microscopy overcame this problem by observing single actin filaments. With TIRF, we now know not only that each end has distinct properties, but also that the rate of γ-phosphate release is much faster from the terminals than from the interior of actin filaments. The rate of γ-phosphate release from actin filament ends is even more accelerated when latrunculin A is bound. These findings highlight the importance of resolving structural differences between actin molecules in the interior of the filament and those at either filament end. This review provides a history of observing actin filaments under light microscopy, an overview of dynamic properties of ATP hydrolysis at the end of actin filament, and structural views of γ-phosphate release.

Structural basis for cofilin binding and actin filament disassembly.

Actin depolymerizing factor (ADF) and cofilin accelerate actin dynamics by severing and disassembling actin filaments. Here, we present the 3.8 Å resolution cryo-EM structure of cofilactin (cofilin-decorated actin filament). The actin subunit structure of cofilactin (C-form) is distinct from those of F-actin (F-form) and monomeric actin (G-form). During the transition between these three conformations, the inner domain of actin (subdomains 3 and 4) and the majority of subdomain 1 move as two separate rigid bodies. The cofilin-actin interface consists of three distinct parts. Based on the rigid body movements of actin and the three cofilin-actin interfaces, we propose models for the cooperative binding of cofilin to actin, preferential binding of cofilin to ADP-bound actin filaments and cofilin-mediated severing of actin filaments.