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.

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.

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.

Novel inter-domain Ca-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 Ca-dependent manner. Two types of evolutionally conserved Ca-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).

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.

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.

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).

Early nucleation events in the polymerization of actin, probed by time-resolved small-angle x-ray scattering.

Nucleators generating new F-actin filaments play important roles in cell activities. Detailed information concerning the events involved in nucleation of actin alone in vitro is fundamental to understanding these processes, but such information has been hard to come by. We addressed the early process of salt-induced polymerization of actin using the time-resolved synchrotron small-angle X-ray scattering (SAXS).

Difference in the hydration water mobility around F-actin and myosin subfragment-1 studied by quasielastic neutron scattering.

Hydration water is essential for a protein to perform its biological function properly. In this study, the dynamics of hydration water around F-actin and myosin subfragment-1 (S1), which are the partner proteins playing a major role in various cellular functions related to cell motility including muscle contraction, was characterized by incoherent quasielastic neutron scattering (QENS). The QENS measurements on the DO- and HO-solution samples of F-actin and S1 provided the spectra of hydration water, from which the translational diffusion coefficient (D), the residence time (τ), and the rotational correlation time (τ) were evaluated.

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.

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.

Structures of the troponin core domain containing the cardiomyopathy-causing mutants studied by small-angle X-ray scattering.

Troponin (Tn), consisting of three subunits, TnC, TnI, and TnT, is a protein in the thin filaments in muscle, and, together with another thin-filament protein tropomyosin (Tm), plays a major role in regulation of muscle contraction. Various mutations of Tn cause familial hypertrophic cardiomyopathy. These mutations are directly related to aberrations in this regulatory mechanism.

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.

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.

Difference in hydration structures between F-actin and myosin subfragment-1 detected by small-angle X-ray and neutron scattering.

Hydration structures around F-actin and myosin subfragment-1 (S1), which play central roles as counterparts in muscle contraction, were investigated by small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS). The radius of gyration of chymotryptic S1 was evaluated to be 41.3±1.

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.

The efficacy and safety of propiverine hydrochloride in patients with overactive bladder symptoms who poorly responded to previous anticholinergic agents.

Objectives. To prospectively examine the efficacy and safety of propiverine hydrochloride in patients with overactive bladder (OAB) symptoms who poorly responded to previous treatment with solifenacin, tolterodine or imidafenacin. Methods.

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.

Human spire interacts with the barbed end of the actin filament.

Spire is an actin nucleator that initiates actin polymerization at a specific place in the cell. Similar to the Arp2/3 complex, spire was initially considered to bind to the pointed end of the actin filament when it generates a new actin filament. Subsequently, spire was reported to be associated with the barbed end (B-end); thus, there is still no consensus regarding the end with which spire interacts.

Electron microscopic visualization of the filament binding mode of actin-binding proteins.

A large number of actin-binding proteins (ABPs) regulate various kinds of cellular events in which the superstructure of the actin cytoskeleton is dynamically changed. Thus, to understand the actin dynamics in the cell, the mechanisms of actin regulation by ABPs must be elucidated. Moreover, it is particularly important to identify the side, barbed-end or pointed-end ABP binding sites on the actin filament.

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.

Structure of hibiscus latent singapore virus by fiber diffraction: a nonconserved his122 contributes to coat protein stability.

Hibiscus latent Singapore virus (HLSV) is a rigid rod-shaped plant virus and a new member of the Tobamovirus family. Unlike all other Tobamoviruses, the HLSV genome contains a unique poly(A) tract in its 3' untranslated region. The virion is composed of a monomeric coat protein (CP) unit of 18 kDa, arranged as a right-handed helix around the virus axis.

Multiple Conformations of F-actin.

Actin works within eukaryotic cells to facilitate a variety of cellular processes, which are driven by the assembly of G-actin (monomeric form) into F-actin (fibrous form), and the disassembly of F-actin into G-actin. F-actin adopts multiple conformations, which are specified by interactions with various actin-binding proteins. Knowledge of the multiple conformations of actin is the key for understanding its cellular functions.

X-ray fiber diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles.

In order to clarify the structural changes of the thin filaments related to the regulation mechanism in skeletal muscle contraction, the intensities of thin filament-based reflections in the X-ray fiber diffraction patterns from live frog skeletal muscles at non-filament overlap length were investigated in the relaxed state and upon activation. Modeling the structural changes of the whole thin filament due to Ca-activation was systematically performed using the crystallographic data of constituent molecules (actin, tropomyosin and troponin core domain) as starting points in order to determine the structural changes of the regulatory proteins and actin. The results showed that the globular core domain of troponin moved toward the filament axis by ∼6 Å and rotated by ∼16° anticlockwise (viewed from the pointed end) around the filament axis by Ca-binding to troponin C, and that tropomyosin together with the tail of troponin T moved azimuthally toward the inner domains of actin by ∼12° and radially by ∼7 Å from the relaxed position possibly to partially open the myosin binding region of actin.

Polymeric structures and dynamic properties of the bacterial actin AlfA.

AlfA is a recently discovered DNA segregation protein from Bacillus subtilis that is distantly related to actin and the bacterial actin homologues ParM and MreB. Here we show that AlfA mostly forms helical 7/3 filaments, with a repeat of about 180 A, that are arranged in three-dimensional bundles. Other polymorphic structures in the form of two-dimensional rafts or paracrystalline nets were also observed.