Catalytic Dwells Research Articles

Eukaryotic yeast V1-ATPase rotary mechanism insights revealed by high-resolution single-molecule studies.

Vacuolar ATP-dependent proton pumps (V-ATPases) belong to a super-family of rotary ATPases and ATP synthases. The V1 complex consumes ATP to drive rotation of a central rotor that pumps protons across membranes via the Vo complex. Eukaryotic V-ATPases are regulated by reversible disassembly of subunit C, V1 without C, and VO. ATP hydrolysis is thought to generate an unknown rotary state that initiates regulated disassembly. Dissociated V1 is inhibited by subunit H that traps it in a specific rotational position. Here, we report the first single-molecule studies with high resolution of time and rotational position of Saccharomyces cerevisiae V1-ATPase lacking subunits H and C (V1ΔHC), which resolves previously elusive dwells and angular velocity changes. Rotation occurred in 120° power strokes separated by dwells comparable to catalytic dwells observed in other rotary ATPases. However, unique V1ΔHC rotational features included: 1) faltering power stroke rotation during the first 60°; 2) a dwell often occurring ∼45° after the catalytic dwell, which did not increase in duration at limiting MgATP; 3) a second dwell, ∼2-fold longer occurring 112° that increased in duration and occurrence at limiting MgATP; 4) limiting MgATP-dependent decreases in power stroke angular velocity where dwells were not observed. The results presented here are consistent with MgATP binding to the empty catalytic site at 112° and MgADP released at ∼45°, and provide important new insight concerning the molecular basis for the differences in rotary positions of substrate binding and product release between V-type and F-type ATPases.

Fast state detection in F1-ATPase rotation enhanced by theory of mixed states and external torque

During brief 120° transitions between long catalytic dwells, single F1-ATPase molecules exhibit angular jumps that vary with rotation angles. Using the angular jump profile enables the detection of fast states in the mechano-chemical scheme of the enzyme, states that are difficult to capture from single-molecule trajectories due to the fluctuations of the imaging nanoprobe. In a previous work, a short-lived, three occupancy state was postulated from a multi-state, probabilistic theory to explain the mean angular jump profile. An assumption in the theory was that the ‘mixing’ of chemical states is negligible during jumps. In a mixing event, two subsequent angular positions recorded by the imaging apparatus belong to two different chemical states of the motor enzyme due to fast reactions within a recording frame. In this paper, we provide an enhanced method for the detection of fast states. On one hand, we show using Langevin simulations that state mixing leads to faster mean angular jump, shifting up the profile. Consequently, the improved method provides a correction to the angular position and lifetime of the postulated three-occupancy metastable state. On the other hand, we show that when F1-ATPase is subject to torques opposing rotation in hydrolysis direction, the torques shift down the dwell angles without affecting the angle-dependent reaction rates. The torques improve detection capability for the fast state by increasing dwell times which is made evident by the flattening of the mean angular jump profile within 40°–60° from the catalytic dwell. In the three-occupancy state release of ADP occurs in concert with the binding of ATP to a different site in the F1-ATPase. Similarly, in the full ATP synthase when torques are created by the proton gradient in the FO region, the release of the product ATP is presumably accelerated by the binding of ADP to a different site in the F1 domain.

The catalytic dwell in ATPases is not crucial for movement against applied torque.

The ATPase-catalysed conversion of ATP to ADP is a fundamental process in biology. During the hydrolysis of ATP, the α3β3 domain undergoes conformational changes while the central stalk (γ/D) rotates unidirectionally. Experimental studies have suggested that different catalytic mechanisms operate depending on the type of ATPase, but the structural and energetic basis of these mechanisms remains unclear. In particular, it is not clear how the positions of the catalytic dwells influence the energy transduction. Here we show that the observed dwell positions, unidirectional rotation and movement against the applied torque are reflections of the free-energy surface of the systems. Instructively, we determine that the dwell positions do not substantially affect the stopping torque. Our results suggest that the three resting states and the pathways that connect them should not be treated equally. The current work demonstrates how the free-energy landscape determines the behaviour of different types of ATPases.

Method to extract multiple states in F1-ATPase rotation experiments from jump distributions

A method is proposed for analyzing fast (10 μs) single-molecule rotation trajectories in F1 adenosinetriphosphatase ([Formula: see text]-ATPase). This method is based on the distribution of jumps in the rotation angle that occur in the transitions during the steps between subsequent catalytic dwells. The method is complementary to the "stalling" technique devised by H. Noji et al. [Biophys. Rev. 9, 103-118, 2017], and can reveal multiple states not directly detectable as steps. A bimodal distribution of jumps is observed at certain angles, due to the system being in either of 2 states at the same rotation angle. In this method, a multistate theory is used that takes into account a viscoelastic fluctuation of the imaging probe. Using an established sequence of 3 specific states, a theoretical profile of angular jumps is predicted, without adjustable parameters, that agrees with experiment for most of the angular range. Agreement can be achieved at all angles by assuming a fourth state with an ∼10 μs lifetime and a dwell angle about 40° after the adenosine 5'-triphosphate (ATP) binding dwell. The latter result suggests that the ATP binding in one β subunit and the adenosine 5'-diphosphate (ADP) release from another β subunit occur via a transient whose lifetime is ∼10 μs and is about 6 orders of magnitude smaller than the lifetime for ADP release from a singly occupied [Formula: see text]-ATPase. An internal consistency test is given by comparing 2 independent ways of obtaining the relaxation time of the probe. They agree and are ∼15 μs.

ATP hydrolysis assists phosphate release and promotes reaction ordering in F1-ATPase.

F1-ATPase (F1) is a rotary motor protein that can efficiently convert chemical energy to mechanical work of rotation via fine coordination of its conformational motions and reaction sequences. Compared with reactant binding and product release, the ATP hydrolysis has relatively little contributions to the torque and chemical energy generation. To scrutinize possible roles of ATP hydrolysis, we investigate the detailed statistics of the catalytic dwells from high-speed single wild-type F1 observations. Here we report a small rotation during the catalytic dwell triggered by the ATP hydrolysis that is indiscernible in previous studies. Moreover, we find in freely rotating F1 that ATP hydrolysis is followed by the release of inorganic phosphate with low synthesis rates. Finally, we propose functional roles of the ATP hydrolysis as a key to kinetically unlock the subsequent phosphate release and promote the correct reaction ordering.

Torque, chemistry and efficiency in molecular motors: a study of the rotary-chemical coupling in F1-ATPase.

Detailed understanding of the action of biological molecular machines must overcome the challenge of gaining a clear knowledge of the corresponding free-energy landscape. An example for this is the elucidation of the nature of converting chemical energy to torque and work in the rotary molecular motor of F1-ATPase. A major part of the challenge involves understanding the rotary–chemical coupling from a non-phenomenological structure/energy description. Here we focused on using a coarse-grained model of F1-ATPase to generate a structure-based free-energy landscape of the rotary–chemical process of the whole system. In particular, we concentrated on exploring the possible impact of the position of the catalytic dwell on the efficiency and torque generation of the molecular machine. It was found that the experimentally observed torque can be reproduced with landscapes that have different positions for the catalytic dwell on the rotary–chemical surface. Thus, although the catalysis is undeniably required for torque generation, the experimentally observed position of the catalytic dwell at 80° might not have a clear advantage for the force generation by F1-ATPase. This further implies that the rotary–chemical couplings in these biological motors are quite robust and their efficiencies do not depend explicitly on the position of the catalytic dwells. Rather, the specific positioning of the dwells with respect to the rotational angle is a characteristic arising due to the structural construct of the molecular machine and might not bear any clear connection to the thermodynamic efficiency for the system.

How release of phosphate from mammalian F1-ATPase generates a rotary substep

The rotation of the central stalk of F1-ATPase is driven by energy derived from the sequential binding of an ATP molecule to its three catalytic sites and the release of the products of hydrolysis. In human F1-ATPase, each 360° rotation consists of three 120° steps composed of substeps of about 65°, 25°, and 30°, with intervening ATP binding, phosphate release, and catalytic dwells, respectively. The F1-ATPase inhibitor protein, IF1, halts the rotary cycle at the catalytic dwell. The human and bovine enzymes are essentially identical, and the structure of bovine F1-ATPase inhibited by IF1 represents the catalytic dwell state. Another structure, described here, of bovine F1-ATPase inhibited by an ATP analog and the phosphate analog, thiophosphate, represents the phosphate binding dwell. Thiophosphate is bound to a site in the α(E)β(E)-catalytic interface, whereas in F1-ATPase inhibited with IF1, the equivalent site is changed subtly and the enzyme is incapable of binding thiophosphate. These two structures provide a molecular mechanism of how phosphate release generates a rotary substep as follows. In the active enzyme, phosphate release from the β(E)-subunit is accompanied by a rearrangement of the structure of its binding site that prevents released phosphate from rebinding. The associated extrusion of a loop in the β(E)-subunit disrupts interactions in the α(E)β(E-)catalytic interface and opens it to its fullest extent. Other rearrangements disrupt interactions between the γ-subunit and the C-terminal domain of the α(E)-subunit. To restore most of these interactions, and to make compensatory new ones, the γ-subunit rotates through 25°-30°.

Anatomy of F 1 -ATPase powered rotation

F1-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank).

Conformational Changes in the β Subunits of F1-Atpase Revealed by Fret Measurements During the Rotation of the γ Subunit

To uncover conformational changes in biomolecules accompanied with biochemical events remains challenging, even though the atomic structures and the kinetics of the biomolecules are revealed. F1-ATPase is a rotary molecular motor in which a central γ subunit rotates against hexagonally arranged subunits α3β3, hydrolyzing ATP sequentially in three β subunits. Previous study using a single-molecule fluorescence polarization method has proposed sets of the β subunit conformations during the rotation (Masaike et al., Nat. Struct. Mol. Biol. 2008). However, further information is indispensable for identifying the ATP-waiting form of which the atomic structure has not been revealed. Here we performed single-pair FRET measurement to detect distance changes between two β subunits. Time trajectories of FRET efficiency showed two-state transitions between high (0.8) and low (0.5). Given the crystal structures, low FRET efficiency indicates one β subunit in the open form but another in the closed form. On the other hand, high FRET efficiency indicates both of the β subunits in the closed form. Next, we performed a simultaneous measurement of FRET between two fluorescently labeled β subunits and the rotation of the γ subunit. High FRET efficiency was occurred in one of the three catalytic dwells. In the remaining five dwells, other two catalytic dwells and three ATP-waiting dwells, FRET efficiency was lower. These results suggest that in the ATP-waiting dwell two of three β subunits would not take the closed form as in the catalytic dwell. We are performing further experiments with a magnetic tweezers.

Binding of Phytopolyphenol Piceatannol Disrupts β/γ Subunit Interactions and Rate-limiting Step of Steady-state Rotational Catalysis in Escherichia coli F1-ATPase

In observations of single molecule behavior under V(max) conditions with minimal load, the F(1) sector of the ATP synthase (F-ATPase) rotates through continuous cycles of catalytic dwells (∼0.2 ms) and 120° rotation steps (∼0.6 ms). We previously established that the rate-limiting transition step occurs during the catalytic dwell at the initiation of the 120° rotation. Here, we use the phytopolyphenol, piceatannol, which binds to a pocket formed by contributions from α and β stator subunits and the carboxyl-terminal region of the rotor γ subunit. Piceatannol did not interfere with the movement through the 120° rotation step, but caused increased duration of the catalytic dwell. The duration time of the intrinsic inhibited state of F(1) also became significantly longer with piceatannol. All of the beads rotated at a lower rate in the presence of saturating piceatannol, indicating that the inhibitor stays bound throughout the rotational catalytic cycle. The Arrhenius plot of the temperature dependence of the reciprocal of the duration of the catalytic dwell (catalytic rate) indicated significantly increased activation energy of the rate-limiting step to trigger the 120° rotation. The activation energy was further increased by combination of piceatannol and substitution of γ subunit Met(23) with Lys, indicating that the inhibitor and the β/γ interface mutation affect the same transition step, even though they perturb physically separated rotor-stator interactions.

The Phytopolyphenol Piceatannol Inhibits the Rate Limiting Step of Rotational Catalysis of the F1-ATPase

Single molecule observations of the F1 sector of the E. coli ATP synthase were made in Vmax conditions with minimal load. The enzyme rotates through continuous cycles of catalytic dwells (pauses lasting ∼0.2 ms) and 120° rotation steps (∼0.6 ms in duration). We previously established that the rate limiting transition state step occurs during the catalytic dwell just prior to the initiation of the 120° rotation. Here we use the phytopolyphenol stilbenoid inhibitor, piceatannol, which binds to a pocket formed by contributions from α and β stator subunits and the carboxyl terminal region of the rotor γ subunit.

Single Molecule Behavior of Inhibited and Active States of Escherichia coli ATP Synthase F1 Rotation

ATP hydrolysis-dependent rotation of the F(1) sector of the ATP synthase is a successive cycle of catalytic dwells (∼0.2 ms at 24 °C) and 120° rotation steps (∼0.6 ms) when observed under V(max) conditions using a low viscous drag 60-nm bead attached to the γ subunit (Sekiya, M., Nakamoto, R. K., Al-Shawi, M. K., Nakanishi-Matsui, M., and Futai, M. (2009) J. Biol. Chem. 284, 22401-22410). During the normal course of observation, the γ subunit pauses in a stochastic manner to a catalytically inhibited state that averages ∼1 s in duration. The rotation behavior with adenosine 5'-O-(3-thiotriphosphate) as the substrate or at a low ATP concentration (4 μM) indicates that the rotation is inhibited at the catalytic dwell when the bound ATP undergoes reversible hydrolysis/synthesis. The temperature dependence of rotation shows that F(1) requires ∼2-fold higher activation energy for the transition from the active to the inhibited state compared with that for normal steady-state rotation during the active state. Addition of superstoichiometric ε subunit, the inhibitor of F(1)-ATPase, decreases the rotation rate and at the same time increases the duration time of the inhibited state. Arrhenius analysis shows that the ε subunit has little effect on the transition between active and inhibited states. Rather, the ε subunit confers lower activation energy of steady-state rotation. These results suggest that the ε subunit plays a role in guiding the enzyme through the proper and efficient catalytic and transport rotational pathway but does not influence the transition to the inhibited state.

1P191 1B1450 ハイブリッドF1-ATPaseの角度解析によって見出されたもうひとつのサブ-サブステップ回転(分子モーター,口頭発表,第48回日本生物物理学会年会)

1P191 1B1450 ハイブリッドF1-ATPaseの角度解析によって見出されたもうひとつのサブ-サブステップ回転(分子モーター,口頭発表,第48回日本生物物理学会年会)

Cooperative Three-step Motions In Catalytic Subunits of F1-ATPase Drive 80° And 40° Substep Rotations

Detecting conformational changes in enzymes is crucial for understanding their functions. Concerning F1-ATPase, the smallest rotary molecular motor ever known, domain motions in three catalytic β-subunits coupled with transitions between chemical states during ATP hydrolysis are assumed to be responsible for rotation of the central shaft γ-subunit. Nevertheless, the extent and timing of motions in β in rotating F1-ATPase still remain largely unknown. Here we directly observe motions in β and rotation of γ simultaneously in the same single molecules; changes in lateral orientation of a single fluorophore fixed to the C-terminal helix of β and substep rotations of beads attached to γ were detected. Furthermore, asymmetric stepping patterns of γ in hybrid F1-ATPase containing one or two mutant β-subunits enabled identification of chemical steps coupled to these motions. The 80° substep of γ is caused by cooperative bending motions of two β-subunits; a ∼40° counterclockwise motion of the first β upon ATP-binding and a clockwise ∼20° motion of the second β upon ADP-release (viewed from Fo side). Meanwhile, the 40°substep of γ is driven by another ∼20° clockwise motion of the third β after ATP-cleavage. Thus three-step cooperative bending of β-subunits causes two substep rotations of γ. Moreover, these results indicate that the initial crystal structure mimics the conformation in the catalytic dwells, and that the conformation in the ATP-waiting dwells contains a novel set of β-subunits; Open, Closed, and partially closed β-subunits. Thus the present study bridges the gap between chemical steps and mechanical work in motor proteins. The present approach to illustrate the enzymatic functions through detection of motions will give insights into mechanisms of diverse enzymes.

Cooperative three-step motions in catalytic subunits of F1-ATPase correlate with 80° and 40° 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.

ATP-driven stepwise rotation of F o F 1 -ATP synthase

FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80 degrees substep rotation, catalytic dwell, and 40 degrees -substep rotation. Compared with F1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATP-driven rotations of FoF1. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized FoF1 and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a Km of approximately 30 microM and a Vmax of approximately 350 revolutions per second (at 37 degrees C). At low ATP, ATP-waiting dwell was seen and the kon-ATP was estimated to be 3.6 x 10(7) M(-1) x s(-1). At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5'-[gamma-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of approximately 80 degrees . The rotational behavior of FoF1 resembles that of F1. This finding indicates that "friction" in Fo motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex.