Direct identification of the rotary angle of ATP cleavage in F1-ATPase from Bacillus PS3.

F1-ATPase is the world's smallest biological rotary motor driven by ATP hydrolysis at three catalytic ß subunits. The 120° rotational step of the central shaft γ consists of 80° substep driven by ATP binding and a subsequent 40° substep. In order to correlate timing of ATP cleavage at a specific catalytic site with a rotary angle, we designed a new F1-ATPase (F1) from thermophilic Bacillus PS3 carrying ß(E190D/F414E/F420E) mutations, which cause extremely slow rates of both ATP cleavage and ATP binding. We produced an F1 molecule that consists of one mutant ß and two wild-type ßs (hybrid F1). As a result, the new hybrid F1 showed two pausing angles that are separated by 200°. They are attributable to two slowed reaction steps in the mutated ß, thus providing the direct evidence that ATP cleavage occurs at 200° rather than 80° subsequent to ATP binding at 0°. This scenario resolves the long-standing unclarified issue in the chemomechanical coupling scheme and gives insights into the mechanism of driving unidirectional rotation.

Characterization of the motility of monomeric kinesin-5/Cin8.

Cin8, the Saccharomyces cerevisiae kinesin-5, has an essential role in mitosis. In in vitro motility assays, tetrameric and dimeric Cin8 constructs showed bidirectional motility in response to ionic strength or Cin8 motor density. However, whether property-switching directionality is present in a monomeric form of Cin8 is unknown. Here we engineered monomeric Cin8 constructs with and without the Cin8-specific ∼99 residues in the loop 8 domain and examined the directionality of these constructs using an in vitro polarity-marked microtubule gliding assay within the range of the motor density or ionic strength. We found that both monomeric constructs showed only plus end-directed activity over the ranges measured, which suggested that minus end-directed motility driven by Cin8 is necessary for at least dimeric forms. Using an in vitro microtubule corkscrewing assay, we also found that monomeric Cin8 corkscrewed microtubules around their longitudinal axes with a constant left-handed pitch. Overall, our results imply that plus-end-directed and left-handed motor activity comprise the intrinsic properties of the Cin8 motor domain as with other monomeric N-kinesins.

Single-molecule pull-out manipulation of the shaft of the rotary motor F1-ATPase.

F1-ATPase is a rotary motor protein in which the central γ-subunit rotates inside the cylinder made of α3ß3 subunits. To investigate interactions between the γ shaft and the cylinder at the molecular scale, load was imposed on γ through a polystyrene bead by three-dimensional optical trapping in the direction along which the shaft penetrates the cylinder. Pull-out event was observed under high-load, and thus load-dependency of lifetime of the interaction was estimated. Notably, accumulated counts of lifetime were comprised of fast and slow components. Both components exponentially dropped with imposed loads, suggesting that the binding energy is compensated by the work done by optical trapping. Because the mutant, in which the half of the shaft was deleted, showed only one fast component in the bond lifetime, the slow component is likely due to the native interaction mode held by multiple interfaces.

Circular orientation fluorescence emitter imaging (COFEI) of rotational motion of motor proteins.

Single-molecule fluorescence polarization technique has been utilized to detect structural changes in biomolecules and intermolecular interactions. Here we developed a single-molecule fluorescence polarization measurement system, named circular orientation fluorescence emitter imaging (COFEI), in which a ring pattern of an acquired fluorescent image (COFEI image) represents an orientation of a polarization and a polarization factor. Rotation and pattern change of the COFEI image allow us to find changes in the polarization by eye and further values of the parameters of a polarization are determined by simple image analysis with high accuracy. We validated its potential applications of COFEI by three assays: 1) Detection of stepwise rotation of F1-ATPase via single quantum nanorod attached to the rotary shaft γ; 2) Visualization of binding of fluorescent ATP analog to the catalytic subunit in F1-ATPase; and 3) Association and dissociation of one head of dimeric kinesin-1 on the microtubule during its processive movement through single bifunctional fluorescent probes attached to the head. These results indicate that the COFEI provides us the advantages of the user-friendly measurement system and persuasive data presentations.

Structural Basis of Backwards Motion in Kinesin-1-Kinesin-14 Chimera: Implication for Kinesin-14 Motility.

Kinesin-14 is a unique minus-end-directed microtubule-based motor. A swinging motion of a class-specific N-terminal neck helix has been proposed to produce minus-end directionality. However, it is unclear how swinging of the neck helix is driven by ATP hydrolysis utilizing the highly conserved catalytic core among all kinesins. Here, using a motility assay, we show that in addition to the neck helix, the conserved five residues at the C-terminal region in kinesin-14, namely the neck mimic, are necessary to give kinesin-1 an ability to reverse its directionality toward the minus end of microtubules. Our structural analyses further demonstrate that the C-terminal neck mimic, in cooperation with conformational changes in the catalytic core during ATP binding, forms a kinesin-14 bundle with the N-terminal neck helix to swing toward the minus end of microtubules. Thus, the neck mimic plays a crucial role in coupling the chemical ATPase reaction with the mechanical cycle to produce the minus-end-directed motility of kinesin-14.

F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements.

Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F1-ATPase (F1) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-α3ß3, while simultaneously monitoring rotations of the central shaft-γ. In the ATP waiting dwell, two of three ß-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two ß-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F1 structure and the rotation angle of the motor. Remarkably, a structure of F1 in an ε-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the αß-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of ß and the loosening/tightening motions at the αß-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F1-ATPase.

Unitary step of gliding machinery in Mycoplasma mobile.

Among the bacteria that glide on substrate surfaces, Mycoplasma mobile is one of the fastest, exhibiting smooth movement with a speed of 2.0-4.5 µm⋅s(-1) with a cycle of attachment to and detachment from sialylated oligosaccharides. To study the gliding mechanism at the molecular level, we applied an assay with a fluorescently labeled and membrane-permeabilized ghost model, and investigated the motility by high precision colocalization microscopy. Under conditions designed to reduce the number of motor interactions on a randomly oriented substrate, ghosts took unitary 70-nm steps in the direction of gliding. Although it remains possible that the stepping behavior is produced by multiple interactions, our data suggest that these steps are produced by a unitary gliding machine that need not move between sites arranged on a cytoskeletal lattice.

Simultaneous observation of the lever arm and head explains myosin VI dual function.

Myosin VI is an adenosine triphosphate (ATP)-driven dimeric molecular motor that has dual function as a vesicle transporter and a cytoskeletal anchor. Recently, it was reported that myosin VI generates three types of steps by taking either a distant binding or adjacent binding state (noncanonical hand-over-hand step pathway). The adjacent binding state, in which both heads bind to an actin filament near one another, is unique to myosin VI and therefore may help explain its distinct features. However, detailed information of the adjacent binding state remains unclear. Here simultaneous observations of the head and tail domain during stepping are presented. These observations show that the lever arms tilt forward in the adjacent binding state. Furthermore, it is revealed that either head could take the subsequent step with equal probability from this state. Together with previous results, a comprehensive stepping scheme is proposed; it includes the tail domain motion to explain how myosin VI achieves its dual function.

A change in the radius of rotation of F1-ATPase indicates a tilting motion of the central shaft.

F(1)-ATPase is a water-soluble portion of F(o)F(1)-ATP synthase and rotary molecular motor that exhibits reversibility in chemical reactions. The rotational motion of the shaft subunit γ has been carefully scrutinized in previous studies, but a tilting motion of the shaft has never been explicitly postulated. Here we found a change in the radius of rotation of the probe attached to the shaft subunit γ between two different intermediate states in ATP hydrolysis: one waiting for ATP binding, and the other waiting for ATP hydrolysis and/or subsequent product release. Analysis of this radial difference indicates a ~4° outward tilting of the γ-subunit induced by ATP binding. The tilt angle is a new parameter, to our knowledge, representing the motion of the γ-subunit and provides a new constraint condition of the ATP-waiting conformation of F(1)-ATPase, which has not been determined as an atomic structure from x-ray crystallography.