Torsional stress in DNA limits collaboration among reverse gyrase molecules.

Reverse gyrase is an enzyme that can overwind (introduce positive supercoils into) DNA using the energy obtained from ATP hydrolysis. The enzyme is found in hyperthermophiles, and the overwinding reaction generally requires a temperature above 70 °C. In a previous study using microscopy, we have shown that 30 consecutive mismatched base pairs (a bubble) in DNA serve as a well-defined substrate site for reverse gyrase, warranting the processive overwinding activity down to 50 °C. Here, we inquire how multiple reverse gyrase molecules may collaborate with each other in overwinding one DNA molecule. We introduced one, two, or four bubbles in a linear DNA that tethered a magnetic bead to a coverslip surface. At 40-71 °C in the presence of reverse gyrase, the bead rotated clockwise as viewed from above, to relax the DNA twisted by reverse gyrase. Dependence on the enzyme concentration indicated that each bubble binds reverse gyrase tightly (dissociation constant < 0.1 nm) and that bound enzyme continuously overwinds DNA for > 5 min. Rotation with two bubbles was significantly faster compared with one bubble, indicating that overwinding actions are basically additive, but four bubbles did not show further acceleration except at 40 °C where the activity was very low. The apparent saturation is due to the hydrodynamic friction against the rotating bead, as confirmed by increasing the medium viscosity. When torsional stress in the DNA, determined by the friction, approaches ~ 7 pN·nm (at 71 °C), the overwinding activity of reverse gyrase drops sharply. Multiple molecules of reverse gyrase collaborate additively within this limit.

Direct observation shows superposition and large scale flexibility within cytoplasmic dynein motors moving along microtubules.

Cytoplasmic dynein is a dimeric AAA(+) motor protein that performs critical roles in eukaryotic cells by moving along microtubules using ATP. Here using cryo-electron microscopy we directly observe the structure of Dictyostelium discoideum dynein dimers on microtubules at near-physiological ATP concentrations. They display remarkable flexibility at a hinge close to the microtubule binding domain (the stalkhead) producing a wide range of head positions. About half the molecules have the two heads separated from one another, with both leading and trailing motors attached to the microtubule. The other half have the two heads and stalks closely superposed in a front-to-back arrangement of the AAA(+) rings, suggesting specific contact between the heads. All stalks point towards the microtubule minus end. Mean stalk angles depend on the separation between their stalkheads, which allows estimation of inter-head tension. These findings provide a structural framework for understanding dynein's directionality and unusual stepping behaviour.

Direct observation of DNA overwinding by reverse gyrase.

Reverse gyrase, found in hyperthermophiles, is the only enzyme known to overwind (introduce positive supercoils into) DNA. The ATP-dependent activity, detected at >70 °C, has so far been studied solely by gel electrophoresis; thus, the reaction dynamics remain obscure. Here, we image the overwinding reaction at 71 °C under a microscope, using DNA containing consecutive 30 mismatched base pairs that serve as a well-defined substrate site. A single reverse gyrase molecule processively winds the DNA for >100 turns. Bound enzyme shows moderate temperature dependence, retaining significant activity down to 50 °C. The unloaded reaction rate at 71 °C exceeds five turns per second, which is >10(2)-fold higher than hitherto indicated but lower than the measured ATPase rate of 20 s(-1), indicating loose coupling. The overwinding reaction sharply slows down as the torsional stress accumulates in DNA and ceases at stress of mere ∼ 5 pN ⋅ nm, where one more turn would cost only sixfold the thermal energy. The enzyme would thus keep DNA in a slightly overwound state to protect, but not overprotect, the genome of hyperthermophiles against thermal melting. Overwinding activity is also highly sensitive to DNA tension, with an effective interaction length exceeding the size of reverse gyrase, implying requirement for slack DNA. All results point to the mechanism where strand passage relying on thermal motions, as in topoisomerase IA, is actively but loosely biased toward overwinding.

A flipped ion pair at the dynein-microtubule interface is critical for dynein motility and ATPase activation.

Dynein is a motor protein that moves on microtubules (MTs) using the energy of adenosine triphosphate (ATP) hydrolysis. To understand its motility mechanism, it is crucial to know how the signal of MT binding is transmitted to the ATPase domain to enhance ATP hydrolysis. However, the molecular basis of signal transmission at the dynein-MT interface remains unclear. Scanning mutagenesis of tubulin identified two residues in α-tubulin, R403 and E416, that are critical for ATPase activation and directional movement of dynein. Electron cryomicroscopy and biochemical analyses revealed that these residues form salt bridges with the residues in the dynein MT-binding domain (MTBD) that work in concert to induce registry change in the stalk coiled coil and activate the ATPase. The R403-E3390 salt bridge functions as a switch for this mechanism because of its reversed charge relative to other residues at the interface. This study unveils the structural basis for coupling between MT binding and ATPase activation and implicates the MTBD in the control of directional movement.

None of the rotor residues of F1-ATPase are essential for torque generation.

F1-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor's high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α3ß3γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α3ß3 subunits using the energy derived from ATP hydrolysis. The rotor consists of an axle, a coiled coil of the amino- and carboxyl-terminal α-helices of γ, which deeply penetrates the stator cylinder, and a globular protrusion that juts out from the stator. Previous work has shown that, for a thermophilic F1, significant portions of the axle can be truncated and the motor still rotates a submicron sized bead duplex, indicating generation of up to half the wild-type (WT) torque. Here, we inquire if any specific interactions between the stator and the rest of the rotor are needed for the generation of a sizable torque. We truncated the protruding portion of the rotor and replaced part of the remaining axle residues such that every residue of the rotor has been deleted or replaced in this or previous truncation mutants. This protrusionless construct showed an unloaded rotary speed about a quarter of the WT, and generated one-third to one-half of the WT torque. No residue-specific interactions are needed for this much performance. F1 is so designed that the basic rotor-stator interactions for torque generation and control of catalysis rely solely upon the shape and size of the rotor at very low resolution. Additional tailored interactions augment the torque to allow ATP synthesis under physiological conditions.

Functions and mechanics of dynein motor proteins.

Fuelled by ATP hydrolysis, dyneins generate force and movement on microtubules in a wealth of biological processes, including ciliary beating, cell division and intracellular transport. The large mass and complexity of dynein motors have made elucidating their mechanisms a sizable task. Yet, through a combination of approaches, including X-ray crystallography, cryo-electron microscopy, single-molecule assays and biochemical experiments, important progress has been made towards understanding how these giant motor proteins work. From these studies, a model for the mechanochemical cycle of dynein is emerging, in which nucleotide-driven flexing motions within the AAA+ ring of dynein alter the affinity of its microtubule-binding stalk and reshape its mechanical element to generate movement.

ATP-driven remodeling of the linker domain in the dynein motor.

Dynein ATPases are the largest known cytoskeletal motors and perform critical functions in cells: carrying cargo along microtubules in the cytoplasm and powering flagellar beating. Dyneins are members of the AAA+ superfamily of ring-shaped enzymes, but how they harness this architecture to produce movement is poorly understood. Here, we have used cryo-EM to determine 3D maps of native flagellar dynein-c and a cytoplasmic dynein motor domain in different nucleotide states. The structures show key sites of conformational change within the AAA+ ring and a large rearrangement of the "linker" domain, involving a hinge near its middle. Analysis of a mutant in which the linker "undocks" from the ring indicates that linker remodeling requires energy that is supplied by interactions with the AAA+ modules. Fitting the dynein-c structures into flagellar tomograms suggests how this mechanism could drive sliding between microtubules, and also has implications for cytoplasmic cargo transport.

The 2.8 Å crystal structure of the dynein motor domain.

Dyneins are microtubule-based AAA(+) motor complexes that power ciliary beating, cell division, cell migration and intracellular transport. Here we report the most complete structure obtained so far, to our knowledge, of the 380-kDa motor domain of Dictyostelium discoideum cytoplasmic dynein at 2.8 Å resolution; the data are reliable enough to discuss the structure and mechanism at the level of individual amino acid residues. Features that can be clearly visualized at this resolution include the coordination of ADP in each of four distinct nucleotide-binding sites in the ring-shaped AAA(+) ATPase unit, a newly identified interaction interface between the ring and mechanical linker, and junctional structures between the ring and microtubule-binding stalk, all of which should be critical for the mechanism of dynein motility. We also identify a long-range allosteric communication pathway between the primary ATPase and the microtubule-binding sites. Our work provides a framework for understanding the mechanism of dynein-based motility.

X-ray structure of a functional full-length dynein motor domain.

Dyneins are large microtubule-based motors that power a wide variety of cellular processes. Here we report a 4.5-Å X-ray crystallographic analysis of the entire functional motor domain of cytoplasmic dynein with ADP from Dictyostelium discoideum, which has revealed the detailed architecture of the functional units required for motor activity, including the ATP-hydrolyzing ring, the long coiled-coil microtubule-binding stalk and the force-generating rod-like linker. We discovered a Y-shaped protrusion composed of two long coiled coils-the stalk and the newly identified 'strut'. This structure supports our model in which the strut coiled coil actively contributes to communication between the primary ATPase site in the ring and the microtubule-binding site at the tip of the stalk coiled coil. Our work also provides insight into how the two motor domains are arranged and how they interact with each other in a functional dimer form of cytoplasmic dynein.

C-sequence of the Dictyostelium cytoplasmic dynein participates in processivity modulation.

We examined the functional roles of C-sequence, a 47-kDa non-AAA+ module at the C-terminal end of the 380-kDa Dictyostelium dynein motor domain. When the distal segment of the C-sequence was deleted from the motor domain, the single-molecule processivity of the dimerized motor domain was selectively impaired without its ensemble motile ability and ATPase activity being severely affected. When the hinge-like sequence between the distal and proximal C-sequence segments was made more or less flexible, the dimeric motor showed lower or higher processivity, respectively. These results suggest a potential function of the distal C-sequence segment as a modulator of processivity.

Biomolecular-motor-based autonomous delivery of lipid vesicles as nano- or microscale reactors on a chip.

We aimed to create an autonomous on-chip system that performs targeted delivery of lipid vesicles (liposomes) as nano- or microscale reactors using machinery from biological systems. Reactor-liposomes would be ideal model cargoes to realize biomolecular-motor-based biochemical analysis chips; however, there are no existing systems that enable targeted delivery of cargo-liposomes in an autonomous manner. By exploiting biomolecular-motor-based motility and DNA hybridization, we demonstrate that single-stranded DNA (ssDNA)-labeled microtubules (MTs), gliding on kinesin-coated surfaces, acted as cargo transporters and that ssDNA-labeled cargo-liposomes were loaded/unloaded onto/from gliding MTs without bursting at loading reservoirs/micropatterned unloading sites specified by DNA base sequences. Our results contribute to the development of an alternative strategy to pressure-driven or electrokinetic flow-based microfluidic devices.

Biomolecular-motor-based nano- or microscale particle translocations on DNA microarrays.

We aimed to create autonomous on-chip systems that perform targeted translocations of nano- or microscale particles in parallel using machinery that mimics biological systems. By exploiting biomolecular-motor-based motility and DNA hybridization, we demonstrate that single-stranded DNA-labeled microtubules gliding on kinesin-coated surfaces acted as cargo translocators and that single-stranded DNA-labeled cargoes were loaded/unloaded onto/from gliding microtubules at micropatterned loading/unloading sites specified by DNA base sequences. Our results will help to create autonomous molecular sorters and sensors.

Helix sliding in the stalk coiled coil of dynein couples ATPase and microtubule binding.

Coupling between ATPase and track binding sites is essential for molecular motors to move along cytoskeletal tracks. In dynein, these sites are separated by a long coiled coil stalk that must mediate communication between them, but the underlying mechanism remains unclear. Here we show that changes in registration between the two helices of the coiled coil can perform this function. We locked the coiled coil at three specific registrations using oxidation to disulfides of paired cysteine residues introduced into the two helices. These trapped ATPase activity either in a microtubule-independent high or low state, and microtubule binding activity either in an ATP-insensitive strong or weak state, depending on the registry of the coiled coil. Our results provide direct evidence that dynein uses sliding between the two helices of the stalk to couple ATPase and microtubule binding activities during its mechanochemical cycle.

AAA+ Ring and linker swing mechanism in the dynein motor.

Dynein ATPases power diverse microtubule-based motilities. Each dynein motor domain comprises a ring-like head containing six AAA+ modules and N- and C-terminal regions, together with a stalk that binds microtubules. How these subdomains are arranged and generate force remains poorly understood. Here, using electron microscopy and image processing of tagged and truncated Dictyostelium cytoplasmic dynein constructs, we show that the heart of the motor is a hexameric ring of AAA+ modules, with the stalk emerging opposite the primary ATPase site (AAA1). The C-terminal region is not an integral part of the ring but spans between AAA6 and near the stalk base. The N-terminal region includes a lever-like linker whose N terminus swings by approximately 17 nm during the ATPase cycle between AAA2 and the stalk base. Together with evidence of stalk tilting, which may communicate changes in microtubule binding affinity, these findings suggest a model for dynein's structure and mechanism.

Protein engineering approaches to study the dynein mechanism using a Dictyostelium expression system.

Dyneins are microtubule-based motor complexes that power a wide variety of motile processes within eukaryotic cells, including the beating of cilia and flagella and intracellular trafficking along microtubules. Mechanistic studies on dynein have been hampered by their enormous size (molecular masses of 0.5-3MDa) and molecular complexity. However, the recent establishment of recombinant expression systems for cytoplasmic dynein, together with structural and functional analyses, has advanced our understanding of the molecular mechanisms of dynein motility. Here, we describe several protocols for protein engineering approaches to the dynein mechanism using a Dictyostelium discoideum expression system. We first describe the design and preparation of recombinant dynein suitable for mechanistic studies. We then discuss two distinct functional assays that take advantage of the recombinant dynein. One is for detection of dynein's conformational changes during the ATPase cycle. Another is an in vitro motility assay at multiple- and single-molecule levels for examination of the dynamic behavior of dynein moving on a microtubule.

BREK/LMTK2 is a myosin VI-binding protein involved in endosomal membrane trafficking.

Myosin VI is involved in a wide range of endocytic and exocytic membrane trafficking pathways; clathrin-mediated endocytosis, intracellular transport of clathrin-coated and -uncoated vesicles, AP-1B-dependent basolateral sorting in polarized epithelial cells and secretion from the Golgi complex to the cell surface. In this study, using a yeast two-hybrid screen, we identified brain-enriched kinase/lemur tyrosine kinase 2 (BREK/LMTK2), a transmembrane serine/threonine kinase with previously unknown cellular functions, as a myosin VI-interacting protein. Several binding experiments confirmed the interaction of myosin VI with BREK in vivo and in vitro. Immunocytochemical analyses revealed that BREK localizes to cytoplasmic membrane vesicles and to perinuclear recycling endosomes. Notably, cells in which BREK was depleted by siRNA were still able to internalize transferrin molecules and to transport them to early endosomes, but were unable to transport them to perinuclear recycling endosomes. Our results show that BREK is critical for the transition of endocytosed membrane vesicles from early endosomes to recycling endosomes and also suggest an involvement of myosin VI in this pathway.

Molecular mechanism of force generation by dynein, a molecular motor belonging to the AAA+ family.

Dynein is an AAA+ (ATPase associated with various cellular activities)-type motor complex that utilizes ATP hydrolysis to actively drive microtubule sliding. The dynein heavy chain (molecular mass >500 kDa) contains six tandemly linked AAA+ modules and exhibits full motor activities. Detailed molecular dissection of this motor with unique architecture was hampered by the lack of an expression system for the recombinant heavy chain, as a result of its large size. However, the recent success of recombinant protein expression with full motor activities has provided a method for advances in structure-function studies in order to elucidate the molecular mechanism of force generation.

Three-dimensional structure of cytoplasmic dynein bound to microtubules.

Cytoplasmic dynein is a large, microtubule-dependent molecular motor (1.2 MDa). Although the structure of dynein by itself has been characterized, its conformation in complex with microtubules is still unknown. Here, we used cryoelectron microscopy (cryo-EM) to visualize the interaction between dynein and microtubules. Most dynein molecules in the nucleotide-free state are bound to the microtubule in a defined conformation and orientation. A 3D image reconstruction revealed that dynein's head domain, formed by a ring-like arrangement of AAA+ domains, is located approximately 280 A away from the center of the microtubule. The order of the AAA+ domains in the ring was determined by using recombinant markers. Furthermore, a 3D helical image reconstruction of microtubules with a dynein's microtubule binding domain [dynein stalk (DS)] revealed that the stalk extends perpendicular to the microtubule. By combining the 3D maps of the dynein-microtubule and DS-microtubule complexes, we present a model for how dynein in the nucleotide-free state binds to microtubules and discuss models for dynein's power stroke.

The coordination of cyclic microtubule association/dissociation and tail swing of cytoplasmic dynein.

The dynein motor domain is composed of a tail, head, and stalk and is thought to generate a force to microtubules by swinging the tail against the head during its ATPase cycle. For this "power stroke," dynein has to coordinate the tail swing with microtubule association/dissociation at the tip of the stalk. Although a detailed picture of the former process is emerging, the latter process remains to be elucidated. By using the single-headed recombinant motor domain of Dictyostelium cytoplasmic dynein, we address the questions of how the interaction of the motor domain with a microtubule is modulated by ATPase steps, how the two mechanical cycles (the microtubule association/dissociation and tail swing) are coordinated, and which ATPase site among the multiple sites in the motor domain regulates the coordination. Based on steady-state and pre-steady-state measurements, we demonstrate that the two mechanical cycles proceed synchronously at most of the intermediate states in the ATPase cycle: the motor domain in the poststroke state binds strongly to the microtubule with a K(d) of approximately 0.2 microM, whereas most of the motor domains in the prestroke state bind weakly to the microtubule with a K(d) of >10 microM. However, our results suggest that the timings of the microtubule affinity change and tail swing are staggered at the recovery stroke step in which the tail swings from the poststroke to the prestroke position. The ATPase site in the AAA1 module of the motor domain was found to be responsible for the coordination of these two mechanical processes.