The nature of the globular- to fibrous-actin transition

120

The actomyosin molecular motor, the motor composed of myosin II and actin filament, is responsible for muscle contraction, converting chemical energy into mechanical work. Although recent single molecule and structural studies have shed new light on the energy-converting mechanism, the physical basis of the molecular-level mechanism remains unclear because of the experimental limitations. To provide a clue to resolve the controversy between the lever-arm mechanism and the Brownian ratchet-like mechanism, we here report an in silico single molecule experiment of an actomyosin motor. When we placed myosin on an actin filament and allowed myosin to move along the filament, we found that myosin exhibits a unidirectional Brownian motion along the filament. This unidirectionality was found to arise from the combination of a nonequilibrium condition realized by coupling to the ATP hydrolysis and a ratchet-like energy landscape inherent in the actin-myosin interaction along the filament, indicating that a Brownian ratchet-like mechanism contributes substantially to the energy conversion of this molecular motor. M olecular machines in living organisms are nanometer-sized small systems working robustly and efficiently in the presence of the severely disturbing thermal noises. Furthermore, in the case of the molecular motors, the energy supplied by the hydrolysis of adenosine triphosphate (ATP), which is to be converted to the mechanical work, is only an order of magnitude larger than the thermal energy at room temperature (∼20k B T), and hence it seems likely that the molecular machines harness the thermal noise, instead of suppressing it as the human-made machines do (1). This distinct feature of the molecular machines should arise from the atomic structures of the constituent proteins, which is reflected in the current focus of interest in the structural aspect of the molecular machines. However, it should be remembered that the structural aspect is not everything but is inseparably linked to the dynamical and energetical aspects. Therefore, a unified description of structure, dynamics, and energetics is the step necessary to understand the molecular-level physical principle of how the molecular machines work.The actomyosin molecular motor, the system composed of myosin II and actin molecules, is responsible for muscle contraction, and is the most extensively studied molecular motor. It has been shown by single molecule experiments (SME) that a single myosin molecule and an actin filament (polymerized actin) constitute the minimal force-generating unit of this motor (2-4) (see Fig. 1A). Although SME has been a powerful approach to study the dynamical aspect of the force-generating unit (2-4), the limitation of the spatiotemporal resolution of SME has inhibited us from developing the full atomistic picture of the force-generating process. On the other hand, since the currently available high-resolution structures of myosin (5-7) are those obtained in the crystalline environment and in the absence of actin, it remains unc...

Section: Methodsmentioning

confidence: 89%

Unidirectional Brownian motion observed in an in silico single molecule experiment of an actomyosin motor

Takano

Terada

Sasai

2010

120

“…We first explored the structural implications of the high divergence of giActin by mapping the Giardia sequence onto a protomer from the Oda et al fibrous (F)-actin structure (9). Of the 155 substitutions between giActin and muscle actin, 48 are highly nonconservative, including six at filament contact points involving both the DNaseI loop (residues 39-47) and hydrophobic plug (residues 266-269) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

Paredez

Assaf

Sept

et al. 2011

166

Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host. cytoskeleton evolution | actin protofilaments | MreB | endocytosis | Rac

“…As starting coordinates for f-actin we used PDB ID code 2ZWH derived from X-ray fiber diffraction data (7). For myosin II we used the crystallographic coordinates 2MYS (8).…”

Section: Resultsmentioning

confidence: 99%

The actin-myosin interface

Lorenz

Holmes

2010

108

165

In order to understand the mechanism of muscle contraction at the atomic level, it is necessary to understand how myosin binds to actin in a reversible way. We have used a novel molecular dynamics technique constrained by an EM map of the actin-myosin complex at 13-Å resolution to obtain an atomic model of the strong-binding (rigor) actin-myosin interface. The constraining force resulting from the EM map during the molecular dynamics simulation was sufficient to convert the myosin head from the initial weak-binding state to the strong-binding (rigor) state. Our actin-myosin model suggests extensive contacts between actin and the myosin head (S1). S1 binds to two actin monomers. The contact surface between actin and S1 has increased dramatically compared with previous models. A number of loops in S1 and actin are involved in establishing the interface. Our model also suggests how the loop carrying the critical Arg 405 Glu mutation in S1 found in a familial cardiomyopathy might be functionally involved.cryoelectron microscopy | myosin II | myosin V T he binding of myosin to actin can be weak or strong. The affinity, which changes over 5 orders of magnitude, is controlled by ATP binding to the myosin head at a position remote from the actin binding site. ATP binding produces "weak binding." In the absence of ATP the binding is "strong." The mechanism of this interaction and its control by ATP is central to an understanding of muscle contraction (1). Thus we need to know the structure of the strong-binding state of myosin to actin in atomic detail. Attempts to crystallize the complex have been unsuccessful. However, incubating myosin heads (subfragment 1, S1) with filamentous actin (f-actin) produces "decorated actin" in which each myosin head binds to the actin filament in the strong-binding or "rigor" configuration. Decorated actin may be used as a model of the actin-myosin "strong-binding" mode. Cryoelectron microscopy and 3D reconstructions of actin decorated with myosin II have produced a density map at 13-Å resolution (2). Combining this with structural data from the head of myosin V, which can be crystallized in the strong-binding myosin conformation (3), gave a near-atomic view of the actin-myosin rigor interaction (4). The actin binding site comprises parts of the upper and lower 50-K domains that are separated by a cleft in the weak-binding form. The cleft closes on strong binding. However, the interface appears incomplete: Some additional local refolding of myosin and actin appears to be necessary. We have now used constrained molecular dynamics-molecular dynamics flexible fitting (MDFF) (5) to fit atomic models into the interface using the EM density map as a constraint. In MDFF the gradient of the density is used as an extra force field. It appears that, in addition to the elements of the upper and lower 50-K domains recognized in the earlier study, part of the flexible loop 2 between the upper and lower 50-K domains in S1 and the actin-DNase I binding loop in actin form essential parts of the interface...