Molecular organization and force-generating mechanism of dynein

Dynein, which is a minus-end-directed microtubule motor, is crucial to a range of cellular processes. The mass of its motor domain is about 10 times that of kinesin, the other microtubule motor. Its large size and the difficulty of expressing and purifying mutants have hampered progress in dynein research. Recently, however, electron microscopy, X-ray crystallography and single-molecule nanometry have shed light on several key unsolved questions concerning how the dynein molecule is organized, what conformational changes in the molecule accompany ATP hydrolysis, and whether two or three motor domains are coordinated in the movements of dynein. This minireview describes our current knowledge of the molecular organization and the force-generating mechanism of dynein, with emphasis on findings from electron microscopy and single-molecule nanometry.     

A rotary molecular motor that can work at near 100% efficiency

A single molecule of F1-ATPase is by itself a rotary motor in which a central gamma-subunit rotates against a surrounding cylinder made of alpha3beta3-subunits. Driven by the three betas that sequentially hydrolyse ATP, the motor rotates in discrete 120 degree steps, as demonstrated in video images of the movement of an actin filament bound, as a marker, to the central gamma-subunit. Over a broad range of load (hydrodynamic friction against the rotating actin filament) and speed, the F1 motor produces a constant torque of ca. 40 pN nm. The work done in a 120 degree step, or the work per ATP molecule, is thus ca. 80 pN nm. In cells, the free energy of ATP hydrolysis is ca. 90 pN nm per ATP molecule, suggesting that the F1 motor can work at near 100% efficiency. We confirmed in vitro that F1 indeed does ca. 80 pN nm of work under the condition where the free energy per ATP is 90 pN nm. The high efficiency may be related to the fully reversible nature of the F1 motor: the ATP synthase, of which F1 is a part, is considered to synthesize ATP from ADP and phosphate by reverse rotation of the F1 motor. Possible mechanisms of F1 rotation are discussed.     

Understanding a molecular motor walking along a microtubule: an asymmetric Brownian motor driven by bubble formation with a focus on binding affinity

In recent years, many studies on a molecular motor have been conducted in the fields of biorheology and nanoengineering. The molecular motor is a molecule that converts the chemical energy obtained by ATP hydrolysis into mechanical energy. Explaining this mechanism is important for nanoengineering. A kinesin, which is a type of molecular motor, has the characteristics to move on a microtubule with hand-over-hand steps. The kinesin walking behaviour is explained by the ‘asymmetric Brownian ratchet model’. Previously, we had suggested that the walking mechanism was achieved by the bubble formation in a nanosized channel surrounded by hydrophobic atoms with the transition between the two states – bubble state and liquid state. However, the walking behaviour of the model motor was different from that of a single molecule measurement of a kinesin. In this study, we constructed a new motor system focused on the asymmetric binding affinity of a motor protein and performed a model simulation using the dissipative particle dynamics method. As a result, it was observed that hand-over-hand walking depends on the transition position ratio and the transition frequency coefficient. Moreover, the efficiency of the new motor system is higher than that of the previous motor systems. The new motor model can provide a simulation guide for the design of biomimetic nanomachines.     

Calcium and cargoes as regulators of myosin 5a activity

Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins.     

A nonmotor microtubule binding site in kinesin-5 is required for filament crosslinking and sliding

Kinesin-5, a widely conserved motor protein required for assembly of the bipolar mitotic spindle in eukaryotes, forms homotetramers with two pairs of motor domains positioned at opposite ends of a dumbbell-shaped molecule [1-3]. It has long been assumed that this configuration of motor domains is the basis of kinesin-5's ability to drive relative sliding of microtubules [2, 4, 5]. Recently, it was suggested that in addition to the N-terminal motor domain, kinesin-5 also has a nonmotor microtubule binding site in its C terminus [6]. However, it is not known how the nonmotor domain contributes to motor activity, or how a kinesin-5 tetramer utilizes a combination of four motor and four nonmotor microtubule binding sites for its microtubule organizing functions. Here we show, in single molecule assays, that kinesin-5 homotetramers require the nonmotor C terminus for crosslinking and relative sliding of two microtubules. Remarkably, this domain enhances kinesin-5's microtubule binding without substantially reducing motor activity. Our?results suggest that tetramerization of kinesin-5's low-processivity motor domains is not sufficient for microtubule sliding because the motor domains alone are unlikely to?maintain persistent microtubule crosslinks. Rather, kinesin-5 utilizes nonmotor microtubule binding sites to tune its microtubule attachment dynamics, enabling it to efficiently align and sort microtubules during metaphase spindle assembly and function.     

The basic mechanism of ATP powered motor proteins

The ability of ATP powered motor proteins to convert chemical free energy into the mechanical work required to move intra-cellular organelles is discussed in terms of the molecular and dynamic fundamentals involved in producing such movements. This is carried out in detail for muscle contraction with the result that in order for a myosin head to act as a motor protein, it is necessary for it to be able to impose a unique series of impacts on an actin filament. It is further shown that these impacts can be generated when a single water molecule is transiently attached to the ADP formed during one step of an ATP cycle in the myosin head. This analysis leads to the conclusion that muscle must be a type of heat machine which has the capability of attaining mechanochemical efficiencies that approach 100%. An extension of ATP powered motor proteins in general is made with the finding that they must share the same motor mechanism of the transiently attached water molecule. A possible application of these considerations to the problem of the active transport of ions is also pointed out.     

Protein-polymer nano-machines. Towards synthetic control of biological processes

The exploitation of nature's machinery at length scales below the dimensions of a cell is an exciting challenge for biologists, chemists and physicists, while advances in our understanding of these biological motifs are now providing an opportunity to develop real single molecule devices for technological applications. Single molecule studies are already well advanced and biological molecular motors are being used to guide the design of nano-scale machines. However, controlling the specific functions of these devices in biological systems under changing conditions is difficult. In this review we describe the principles underlying the development of a molecular motor with numerous potential applications in nanotechnology and the use of specific synthetic polymers as prototypic molecular switches for control of the motor function. The molecular motor is a derivative of a TypeI Restriction-Modification (R-M) enzyme and the synthetic polymer is drawn from the class of materials that exhibit a temperature-dependent phase transition.The potential exploitation of single molecules as functional devices has been heralded as the dawn of new era in biotechnology and medicine. It is not surprising, therefore, that the efforts of numerous multidisciplinary teams 12. have been focused in attempts to develop these systems. as machines capable of functioning at the low sub-micron and nanometre length-scales 3. However, one of the obstacles for the practical application of single molecule devices is the lack of functional control methods in biological media, under changing conditions. In this review we describe the conceptual basis for a molecular motor (a derivative of a TypeI Restriction-Modification enzyme) with numerous potential applications in nanotechnology and the use of specific synthetic polymers as prototypic molecular switches for controlling the motor function 4.     

Molecular processes of inhibition and stimulation of ATP synthase caused by the phytotoxin tentoxin

F(1)-ATPase is the smallest mechanical motor known. Tentoxin, a cyclic peptide produced by phytopathogenic fungi, inactivates the F(1) motor in sensitive plants at nanomolar to micromolar concentrations, whereas higher concentrations surpass the natural activity of the enzyme. Single molecule studies now have clarified the molecular steps involved in both processes. Inactivation delays the dwell time of a single step in the complete 360 degrees turn and results in an asymmetric rotation of the central rotor subunit. In contrast, rotation in the stimulated F(1) particle is smooth and accompanied by strongly reduced ADP inhibition. Our study provides for the first time the direct observation of a noncompetitively inhibited state of the enzyme and directly visualizes the regulation of the molecular motor by an external natural compound. In addition, the ADP release step during catalysis was revealed by analysis of the single molecule rotation behavior. Hence, tentoxin is a sophisticated molecular tool to mark and control certain catalytic steps within the reaction pathway of the molecular F(1) motor.     

Regulation of the function of mammalian myosin and its conformational change

It has been known that the phosphorylation of the regulatory light chain, residing at the head/rod junction of the molecule activates the motor activity of smooth muscle and non-muscle conventional myosin (myosin II), and triggers a large conformational change of the molecule from the inhibited folded conformation to the active extended conformation. Recent structural analysis has revealed the structural basis of the inhibition of the motor function of the two heads in the inhibited conformation. On the other hand, recent studies have revealed that a processive unconventional myosin, myosin V, also shows a large change in the conformation from the folded to an extended form and this explains the activation mechanism of myosin V motor activity. These findings suggest the presence of a common scenario for the regulation of motor protein functions.     

Use of negative stain and single-particle image processing to explore dynamic properties of flexible macromolecules

Flexible macromolecules pose special difficulties for structure determination by crystallography or NMR. Progress can be made by electron microscopy, but electron cryo-microscopy of unstained, hydrated specimens is limited to larger macromolecules because of the inherently low signal-to-noise ratio. For three-dimensional structure determination, the single particles must be invariant in structure. Here, we describe how we have used negative staining and single-particle image processing techniques to explore the structure and flexibility of single molecules of two motor proteins: myosin and dynein. Critical for the success of negative staining is a hydrophilic, thin carbon film, because it produces a low noise background around each molecule, and stabilises the molecule against damage by the stain. The strategy adopted for single-particle image processing exploits the flexibility available within the SPIDER software suite. We illustrate the benefits of successive rounds of image alignment and classification, and the use of whole molecule averages and movies to analyse and display both structure and flexibility within the dynein motor.     

DNA mechanics as a tool to probe helicase and translocase activity

Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling.     

A cytoplasmic dynein tail mutation impairs motor processivity

Mutations in the tail of the cytoplasmic dynein molecule have been reported to cause neurodegenerative disease in mice. The mutant mouse strain Legs at odd angles (Loa) has impaired retrograde axonal transport, but the molecular deficiencies in the mutant dynein molecule, and how they contribute to neurodegeneration, are unknown. To address these questions, we purified dynein from wild-type mice and the Legs at odd angles mutant mice. Using biochemical, single-molecule, and live-cell-imaging techniques, we find a marked inhibition of motor run-length in vitro and in vivo, and significantly altered motor domain coordination in the dynein from mutant mice. These results suggest a potential role for the dynein tail in motor function, and provide direct evidence for a link between single-motor processivity and disease.     

Single molecule thermodynamics in biological motors

Biological molecular machines use thermal activation energy to carry out various functions. The process of thermal activation has the stochastic nature of output events that can be described according to the laws of thermodynamics. Recently developed single molecule detection techniques have allowed each distinct enzymatic event of single biological machines to be characterized providing clues to the underlying thermodynamics. In this study, the thermodynamic properties in the stepping movement of a biological molecular motor have been examined. A single molecule detection technique was used to measure the stepping movements at various loads and temperatures and a range of thermodynamic parameters associated with the production of each forward and backward step including free energy, enthalpy, entropy and characteristic distance were obtained. The results show that an asymmetry in entropy is a primary factor that controls the direction in which the motor will step. The investigation on single molecule thermodynamics has the potential to reveal dynamic properties underlying the mechanisms of how biological molecular machines work.     

Working strokes by single molecules of the kinesin-related microtubule motor ncd

The ncd protein is a dimeric, ATP-powered motor that belongs to the kinesin family of microtubule motor proteins. Here we resolve single mechanochemical cycles of recombinant, dimeric, full-length ncd, using optical-tweezers-based instrumentation and a three-bead, suspended-microtubule assay. Under conditions of limiting ATP, isolated and transient microtubule-binding events exhibit exponentially distributed and ATP-concentration-dependent lifetimes. These events do not involve consecutive steps along the microtubule, quantitatively confirming that ncd is non-processive. At low loads, a single motor molecule produces ATP-triggered working strokes of about 9 nm, which occur at the ends of binding events.     

A model for the oscillatory motion of single dynein molecules

Axonemal dynein is the molecular motor responsible for the rhythmic beating of eukaryotic cilia and flagella. An individual axonemal dynein molecule is capable of both unidirectional and oscillatory motion along a microtubule (Nature 393 (1998) 711). We propose a model which links the physical motion of a two-headed dynein molecule to its ATP hydrolysis cycle, and which exhibits both processive and oscillatory behaviors. A mathematical analysis of the model is used to make experimentally testable predictions.     

Motor potential profile and a robust method for extracting it from time series of motor positions

Molecular motors are small, and, as a result, motor operation is dominated by high-viscous friction and large thermal fluctuations from the surrounding fluid environment. The small size has hindered, in many ways, the studies of physical mechanisms of molecular motors. For a macroscopic motor, it is possible to observe/record experimentally the internal operation details of the motor. This is not yet possible for molecular motors. The chemical reaction in a molecular motor has many occupancy states, each having a different effect on the motor motion. The overall effect of the chemical reaction on the motor motion can be characterized by the motor potential profile. The potential profile reveals how the motor force changes with position in a motor step, which may lead to insights into how the chemical reaction is coupled to force generation. In this article, we propose a mathematical formulation and a robust method for constructing motor potential profiles from time series of motor positions measured in single molecule experiments. Numerical examples based on simulated data are shown to demonstrate the method. Interestingly, it is the small size of molecular motors (negligible inertia) that makes it possible to recover the potential profile from time series of motor positions. For a macroscopic motor, the variation of driving force within a cycle is smoothed out by the large inertia.     

Converting a motor to an anchor

Myosin VI can move along actin filaments to serve as a transport motor. It is also thought to anchor vesicles or proteins to actin. How these two diverse activities, which require very different modes of interaction with actin, are mediated is not understood. Using single molecule observations, Altman et al. (2004 [this issue of Cell]) demonstrate that load applied to myosin VI can convert this motor from a transporter to an anchor.     

Models of bacteriophage DNA packaging motors

An ATP-dependent motor drives a DNA genome into a bacteriophage capsid during morphogenesis of double-stranded DNA bacteriophages both in vivo and in vitro. The DNA molecule enters the capsid through a channel in the center of a symmetric protein ring called a connector. Mechanisms in two classes have been proposed for this motor: (1) An ATP-driven rotating connector pulls a DNA molecule via serial power strokes. (2) The connector rectifies DNA motion that is either thermal, biased thermal, or oscillating electrical field-induced (motor-ratchet hypothesis). Mechanisms in the first class have previously been proposed to explain the detailed structure of DNA packaging motors. The present study demonstrates that the motor-ratchet hypothesis also explains the current data, including data in the following categories: biochemical genetics, energetics, structure, and packaging dynamics.