Functional and structural analysis of a cyclization domain in a cyclic ß-1,2-glucan synthase.

Cyclic ß-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic ß-1,2-glucans (CßGs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that ß-glucosidase-resistant compounds are produced by TiCGSCy with linear ß-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are CßGs. Next, action pattern analyses using ß-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide being the minimum length of the substrate. These analyses also showed that longer substrate ß-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce CßGs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of ß-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear differences in the reaction pathways between these enzymes. Overall, we propose a novel reaction mechanism of TiCGSCy. Thus, the present group of CGSs defines a new glycoside hydrolase family, GH189. KEY POINTS: • It was clearly evidenced that cyclization domain alone produces cyclic ß-1,2-glucans. • The domain exclusively catalyzes transglycosylation without hydrolysis. • The present catalytic domain defines as a new glycoside hydrolase family 189.

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.

Insights into the mechanism of ATP-driven rotary motors from direct torque measurement.

Motor proteins are molecular machines that convert chemical energy into mechanical work. In addition to existing studies performed on the linear motors found in eukaryotic cells, researchers in biophysics have also focused on rotary motors such as F1-ATPase. Detailed studies on the rotary F1-ATPase motor have correlated all chemical states to specific mechanical events at the single-molecule level. Recent studies showed that there exists another ATP-driven protein motor in life: the rotary machinery that rotates archaeal flagella (archaella). Rotation speed, stepwise movement, and variable directionality of the motor of Halobacterium salinarum were described in previous studies. Here we review recent experimental work discerning the molecular mechanism underlying how the archaellar motor protein FlaI drives rotation by generation of motor torque. In combination, those studies found that rotation slows as the viscous drag of markers increases, but torque remains constant at 160 pN·nm independent of rotation speed. Unexpectedly, the estimated work done in a single rotation is twice the expected energy that would come from hydrolysis of six ATP molecules in the FlaI hexamer. To reconcile the apparent contradiction, a new and general model for the mechanism of ATP-driven rotary motors is discussed.

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.

Three-dimensional tracking of microbeads attached to the tip of single isolated tracheal cilia beating under external load.

To study the properties of tracheal cilia beating under various conditions, we developed a method to monitor the movement of the ciliary tip. One end of a demembranated cilium was immobilized on the glass surface, while the other end was capped with a polystyrene bead and tracked in three dimensions. The cilium, when activated by ATP, stably repeated asymmetric beating as in vivo. The tip of a cilium in effective and recovery strokes moved in discrete trajectories that differed in height. The trajectory remained asymmetric in highly viscous solutions. Model calculation showed that cilia maintained a constant net flux during one beat cycle irrespective of the medium viscosity. When the bead attached to the end was trapped with optical tweezers, it came to display linear oscillation only in the longitudinal direction. Such a beating-mode transition may be an inherent nature of movement-restricted cilia.

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.

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.

Thermodynamic analyses of nucleotide binding to an isolated monomeric ß subunit and the α3ß3γ subcomplex of F1-ATPase.

Rotation of the γ subunit of the F1-ATPase plays an essential role in energy transduction by F1-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F1-ATPase. In this study, we introduced a ßY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a ßE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric ßY341W subunit and a mutant α3ß3γ subcomplex containing the ßY341W mutation with or without an additional ßE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/Kd) of the nucleotides for the isolated ß subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van't Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated ß subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH(0) and ΔG(0) values at 25°C. The binding of nucleotides with different bases to the isolated ß subunit resulted in different entropy changes. Interestingly, NDP binding to the α3ß(Y341W)3γ subcomplex had similar Kd and ΔG(0) values as binding to the isolated ß(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.

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.

Simultaneous observation of chemomechanical coupling of a molecular motor.

F(1)-ATPase is the smallest rotary molecular motor ever found. Unidirectional rotation of the γ-shaft is driven by precisely coordinated sequential ATP hydrolysis reactions in three catalytic sites arranged 120° apart in the cylinder. Single-molecule observation allows us to directly watch the rotation of the shaft using micron-sized plastic beads. Additionally, an advanced version of "total internal reflection fluorescence microscope (TIRFM)" enables us to detect binding and release of energy currency through fluorescently labeled ATP. In this chapter, we describe how to set up the system for simultaneous observation of these two critical events. This specialized optical setup is applicable to a variety of research, not only molecular motors but also other single-molecule topics.

Cooperative three-step motions in catalytic subunits of F(1)-ATPase correlate with 80 degrees and 40 degrees substep rotations.

Rotation of the central shaft gamma subunit in a molecular motor F(1)-ATPase is assumed to correlate with and probably be driven by domain motions of the three catalytic beta subunits. Here we observe directly these beta motions through an attached fluorophore, concomitantly with 80 degrees and 40 degrees substep rotations of gamma in the same single molecules. We show the sequence of conformations that each beta subunit undergoes in three-step bending, a approximately 40 degrees counterclockwise turn followed by two approximately 20 degrees clockwise turns, occurring in synchronization with two substep rotations of gamma. The results indicate that most previous crystal structures mimic the conformational set of three beta subunits in the catalytic dwells. Moreover, a previously undescribed set of beta conformations, open, closed and partially closed, is revealed in the ATP-waiting dwells. The present study thus bridges the gap between the chemical and mechanical steps in F(1)-ATPase.

Single-molecule observation of rotation of F1-ATPase through microbeads.

F(o)F(1)-ATP synthase catalyzes the synthesis of ATP using proton-motive force across a membrane. When isolated, the F1 sector, composed of five polypeptide chains with a stoichiometry of alpha(3)beta(3)gammadeltaepsilon, solely hydrolyzes ATP into ADP and phosphate, and is thus called F(1)-ATPase. Rotation of a shaft domain in F(o)F(1)-ATP synthase has been hypothesized by Paul Boyer, and ultimately was confirmed by direct observation as rotation of the gamma-subunit in an isolated alpha(3)beta(3)gamma subcomplex. Unitary turnover of ATP induces 120 degrees steps, consistent with the configuration of three catalytic sites arranged 120 degrees apart around gamma. We have shown the relationships between chemical and mechanical events by imaging individual F(1) molecules under an optical microscope. A new scheme emerges: as soon as a catalytic site binds ATP, the gamma-subunit always turns the same face (interaction surface) to the beta hosting that site; approximately 80 degrees rotation is driven by ATP binding; approximately 40 degrees rotation is induced by completion of hydrolysis [and/or phosphate release] in the site that bound ATP one step earlier.

Probing conformations of the beta subunit of F0F1-ATP synthase in catalysis.

A subcomplex of F0F1-ATP synthase (F0F1), alpha3beta3gamma, was shown to undergo the conformation(s) during ATP hydrolysis in which two of the three beta subunits have the "Closed" conformation simultaneously (CC conformation) [S.P. Tsunoda, E. Muneyuki, T. Amano, M. Yoshida, H. Noji, Cross-linking of two beta subunits in the closed conformation in F1-ATPase, J. Biol. Chem. 274 (1999) 5701-5706]. This was examined by the inter-subunit disulfide cross-linking between two mutant beta(I386C)s that was formed readily only when the enzyme was in the CC conformation. Here, we adopted the same method for the holoenzyme F0F1 from Bacillus PS3 and found that the CC conformation was generated during ATP hydrolysis but barely during ATP synthesis. The experiments using F0F1 with the epsilon subunit lacking C-terminal helices further suggest that this difference is related to dynamic nature of the epsilon subunit and that ATP synthesis is accelerated when it takes the pathway involving the CC conformation.

The ATP-waiting conformation of rotating F1-ATPase revealed by single-pair fluorescence resonance energy transfer.

F1-ATPase is an ATP-driven rotary motor in which a rod-shaped gamma subunit rotates inside a cylinder made of alpha3beta3 subunits. To elucidate the conformations of rotating F1, we measured fluorescence resonance energy transfer (FRET) between a donor on one of the three betas and an acceptor on gamma in single F1 molecules. The yield of FRET changed stepwise at low ATP concentrations, reflecting the stepwise rotation of gamma. In the ATP-waiting state, the FRET yields indicated a gamma position approximately 40 degrees counterclockwise (= direction of rotation) from that in the crystal structures of mitochondrial F1, suggesting that the crystal structures mimic a metastable state before product release.

Stepping rotation of F(1)-ATPase with one, two, or three altered catalytic sites that bind ATP only slowly.

F(1)-ATPase is an ATP hydrolysis-driven motor in which the gamma subunit rotates in the stator cylinder alpha(3)beta(3). To know the coordination of three catalytic beta subunits during catalysis, hybrid F(1)-ATPases, each containing one, two, or three "slow" mutant beta subunits that bind ATP very slowly, were prepared, and the rotations were observed with a single molecule level. Each hybrid made one, two, or three steps per 360 degrees revolution, respectively, at 5 microm ATP where the wild-type enzyme rotated continuously without step under the same observing conditions. The observed dwell times of the steps are explained by the slow binding rate of ATP. Except for the steps, properties of rotation, such as the torque forces exerted during rotary movement, were not significantly changed from those of the wild-type enzyme. Thus, it appears that the presence of the slow beta subunit(s) does not seriously affect other normal beta subunit(s) in the same F(1)-ATPase molecule and that the order of sequential catalytic events is faithfully maintained even when ATP binding to one or two of the catalytic sites is retarded.

F1-ATPase changes its conformations upon phosphate release.

Motor proteins, myosin, and kinesin have gamma-phosphate sensors in the switch II loop that play key roles in conformational changes that support motility. Here we report that a rotary motor, F1-ATPase, also changes its conformations upon phosphate release. The tryptophan mutation was introduced into Arg-333 in the beta subunit of F1-ATPase from thermophilic Bacillus PS3 as a probe of conformational changes. This residue interacts with the switch II loop (residues 308-315) of the beta subunit in a nucleotide-bound conformation. The addition of ATP to the mutant F1 subcomplex alpha3beta(R333W)3gamma caused transient increase and subsequent decay of the Trp fluorescence. The increase was caused by conformational changes on ATP binding. The rate of decay agreed well with that of phosphate release monitored by phosphate-binding protein assays. This is the first evidence that the beta subunit changes its conformation upon phosphate release, which may share a common mechanism of exerting motility with other motor proteins.