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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.

We aim to develop a theory based on a concept other than the chemo-mechanical coupling (transduction of chemical free energy of ATP to mechanical work) for an ATP-driven protein complex. Experimental results conflicting with the chemo-mechanical coupling have recently emerged. We claim that the system comprises not only the protein complex but also the aqueous solution in which the protein complex is immersed and the system performs essentially no mechanical work. We perform statistical-mechanical analyses on V1-ATPase (the A3B3DF complex) for which crystal structures in more different states are experimentally known than for F1-ATPase (the α3β3γ complex). Molecular and atomistic models are employed for water and the structure of V1-ATPase, respectively. The entropy originating from the translational displacement of water molecules in the system is treated as a pivotal factor. We find that the packing structure of the catalytic dwell state of V1-ATPase is constructed by the interplay of ATP bindings to two of the A subunits and incorporation of the DF subunit. The packing structure represents the nonuniformity with respect to the closeness of packing of the atoms in constituent proteins and protein interfaces. The physical picture of rotation mechanism of F1-ATPase recently constructed by Kinoshita is examined, and common points and differences between F1- and V1-ATPases are revealed. An ATP hydrolysis cycle comprises binding of ATP to the protein complex, hydrolysis of ATP into ADP and Pi in it, and dissociation of ADP and Pi from it. During each cycle, the chemical compounds bound to the three A or β subunits and the packing structure of the A3B3 or α3β3 complex are sequentially changed, which induces the unidirectional rotation of the central shaft for retaining the packing structure of the A3B3DF or α3β3γ complex stabilized for almost maximizing the water entropy. The torque driving the rotation is generated by water with no input of chemical free energy. The presence of ATP is indispensable as a trigger of the torque generation. The ATP hydrolysis or synthesis reaction is tightly coupled to the rotation of the central shaft in the normal or inverse direction through the water-entropy effect.

The F-ATP synthase, consisting of F1 and FO motors connected by a central rotor and the stators, is the enzyme responsible for synthesizing the majority of ATP in all organisms. The F1 (αβ)3 ring stator contains three catalytic sites. Single-molecule F1 rotation studies revealed that ATP hydrolysis at each catalytic site (0°) precedes a power-stroke that rotates subunit-γ 120° with angular velocities that vary with rotational position. Catalytic site conformations vary relative to subunit-γ position (βE, empty; βD, ADP bound; βT, ATP-bound). During a power stroke, βE binds ATP (0°–60°) and βD releases ADP (60°–120°). Årrhenius analysis of the power stroke revealed that elastic energy powers rotation via unwinding the γ-subunit coiled-coil. Energy from ATP binding at 34° closes βE upon subunit-γ to drive rotation to 120° and forcing the subunit-γ to exchange its tether from βE to βD, which changes catalytic site conformations. In F1FO, the membrane-bound FO complex contains a ring of c-subunits that is attached to subunit-γ. This c-ring rotates relative to the subunit-a stator in response to transmembrane proton flow driven by a pH gradient, which drives subunit-γ rotation in the opposite direction to force ATP synthesis in F1. Single-molecule studies of F1FO embedded in lipid bilayer nanodisks showed that the c-ring transiently stopped F1-ATPase-driven rotation every 36° (at each c-subunit in the c10-ring of E. coli F1FO) and was able to rotate 11° in the direction of ATP synthesis. Protonation and deprotonation of the conserved carboxyl group on each c-subunit is facilitated by separate groups of subunit-a residues, which were determined to have different pKa’s. Mutations of any of any residue from either group changed both pKa values, which changed the occurrence of the 11° rotation proportionately. This supports a Grotthuss mechanism for proton translocation and indicates that proton translocation occurs during the 11° steps. This is consistent with a mechanism in which each 36° of rotation the c-ring during ATP synthesis involves a proton translocation-dependent 11° rotation of the c-ring, followed by a 25° rotation driven by electrostatic interaction of the negatively charged unprotonated carboxyl group to the positively charged essential arginine in subunit-a. [ABSTRACT FROM AUTHOR]

The F-ATP synthase, consisting of F1 and FO motors connected by a central rotor and the stators, is the enzyme responsible for synthesizing the majority of ATP in all organisms. The F1 (αβ)3 ring stator contains three catalytic sites. Single-molecule F1 rotation studies revealed that ATP hydrolysis at each catalytic site (0°) precedes a power-stroke that rotates subunit-γ 120° with angular velocities that vary with rotational position. Catalytic site conformations vary relative to subunit-γ position (βE, empty; βD, ADP bound; βT, ATP-bound). During a power stroke, βE binds ATP (0°–60°) and βD releases ADP (60°–120°). Årrhenius analysis of the power stroke revealed that elastic energy powers rotation via unwinding the γ-subunit coiled-coil. Energy from ATP binding at 34° closes βE upon subunit-γ to drive rotation to 120° and forcing the subunit-γ to exchange its tether from βE to βD, which changes catalytic site conformations. In F1FO, the membrane-bound FO complex contains a ring of c-subunits that is attached to subunit-γ. This c-ring rotates relative to the subunit-a stator in response to transmembrane proton flow driven by a pH gradient, which drives subunit-γ rotation in the opposite direction to force ATP synthesis in F1. Single-molecule studies of F1FO embedded in lipid bilayer nanodisks showed that the c-ring transiently stopped F1-ATPase-driven rotation every 36° (at each c-subunit in the c10-ring of E. coli F1FO) and was able to rotate 11° in the direction of ATP synthesis. Protonation and deprotonation of the conserved carboxyl group on each c-subunit is facilitated by separate groups of subunit-a residues, which were determined to have different pKa’s. Mutations of any of any residue from either group changed both pKa values, which changed the occurrence of the 11° rotation proportionately. This supports a Grotthuss mechanism for proton translocation and indicates that proton translocation occurs during the 11° steps. This is consistent with a mechanism in which each 36° of rotation the c-ring during ATP synthesis involves a proton translocation-dependent 11° rotation of the c-ring, followed by a 25° rotation driven by electrostatic interaction of the negatively charged unprotonated carboxyl group to the positively charged essential arginine in subunit-a.

ATPase inhibitory factor 1 (IF1) is a mitochondrial regulatory protein that blocks ATP hydrolysis of F1-ATPase, by inserting its N -terminus into the rotor–stator interface of F1-ATPase. Although previous studies have proposed a two-step model for IF1-mediated inhibition, the underlying molecular mechanism remains unclear. Here, we analysed the kinetics of IF1-mediated inhibition under a wide range of [ATP]s and [IF1]s, using bovine mitochondrial IF1 and F1-ATPase. Typical hyperbolic curves of inhibition rates with [IF1]s were observed at all [ATP]s tested, suggesting a two-step mechanism: the initial association of IF1 to F1-ATPase and the locking process, where IF1 blocks rotation by inserting its N -terminus. The initial association was dependent on ATP. Considering two principal rotation dwells, binding dwell and catalytic dwell, in F1-ATPase, this result means that IF1 associates with F1-ATPase in the catalytic-waiting state. In contrast, the isomerization process to the locking state was almost independent of ATP, suggesting that it is also independent of the F1-ATPase state. Further, we investigated the role of Glu30 or Tyr33 of IF1 in the two-step mechanism. Kinetic analysis showed that Glu30 is involved in the isomerization, whereas Tyr33 contributes to the initial association. Based on these findings, we propose an IF1-mediated inhibition scheme. [ABSTRACT FROM AUTHOR]

The rotary bacterial flagellar motor is remarkable in biochemistry for its highly synchronized operation and amplification during switching of rotation sense. The motor is part of the flagellar basal body, a complex multi-protein assembly. Sensory and energy transduction depends on a core of six proteins that are adapted in different species to adjust torque and produce diverse switches. Motor response to chemotactic and environmental stimuli is driven by interactions of the core with small signal proteins. The initial protein interactions are propagated across a multi-subunit cytoplasmic ring to switch torque. Torque reversal triggers structural transitions in the flagellar filament to change motile behavior. Subtle variations in the core components invert or block switch operation. The mechanics of the flagellar switch have been studied with multiple approaches, from protein dynamics to single molecule and cell biophysics. The architecture, driven by recent advances in electron cryo-microscopy, is available for several species. Computational methods have correlated structure with genetic and biochemical databases. The design principles underlying the basis of switch ultra-sensitivity and its dependence on motor torque remain elusive, but tantalizing clues have emerged. This review aims to consolidate recent knowledge into a unified platform that can inspire new research strategies.

F1- and V1-ATPase are rotary molecular motors that convert chemical energy released upon ATP hydrolysis into torque to rotate a central rotor axle against the surrounding catalytic stator cylinder with high efficiency. How conformational change occurring in the stator is coupled to the rotary motion of the axle is the key unknown in the mechanism of rotary motors. Here, we generated chimeric motor proteins by inserting an exogenous rod protein, FliJ, into the stator ring of F1 or of V1 and tested the rotation properties of these chimeric motors. Both motors showed unidirectional and continuous rotation, despite no obvious homology in amino acid sequence between FliJ and the intrinsic rotor subunit of F1 or V1. These results showed that any residue-specific interactions between the stator and rotor are not a prerequisite for unidirectional rotation of both F1 and V1. The torque of chimeric motors estimated from viscous friction of the rotation probe against medium revealed that whereas the F1-FliJ chimera generates only 10% of WT F1, the V1-FliJ chimera generates torque comparable to that of V1 with the native axle protein that is structurally more similar to FliJ than the native rotor of F1. This suggests that the gross structural mismatch hinders smooth rotation of FliJ accompanied with the stator ring of F1. [ABSTRACT FROM AUTHOR]

Abstract: F1-ATPase catalyzes ATP hydrolysis to drive the central γ-shaft rotating inside a hexameric cylinder composed of alternating α and β subunits. Experiments showed that the rotation of γ-shaft proceeds in steps of 120° and each 120°-rotation is composed of an 80° substep and a 40° substep. Here, based on the previously proposed models, an improved physical model for chemomechanical coupling of F1-ATPase is presented, with which the two-substep rotation is well explained. One substep is driven by the power stroke upon ATP binding, while the other one resulted from the passage of γ-shaft from previous to next adjacent β subunits via free diffusion. Using the model, the dynamics and kinetics of F1-ATPase, such as the rotating time of each substep, the dwell time at each pause and the rotation rate, are analytically studied. The theoretical results obtained with only three adjustable parameters reproduce the available experimental data well. [Copyright &y& Elsevier]

Abstract: Archaeal ATP synthase (A-ATPase) is the functional homolog to the ATP synthase found in bacteria, mitochondria and chloroplasts, but the enzyme is structurally more related to the proton-pumping vacuolar ATPase found in the endomembrane system of eukaryotes. We have cloned, overexpressed and characterized the stator-forming subunits E and H of the A-ATPase from the thermoacidophilic Archaeon, Thermoplasma acidophilum. Size exclusion chromatography, CD, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and NMR spectroscopic experiments indicate that both polypeptides have a tendency to form dimers and higher oligomers in solution. However, when expressed together or reconstituted, the two individual polypeptides interact with high affinity to form a stable heterodimer. Analyses by gel filtration chromatography and analytical ultracentrifugation show the heterodimer to have an elongated shape, and the preparation to be monodisperse. Thermal denaturation analyses by CD and differential scanning calorimetry revealed the more cooperative unfolding transitions of the heterodimer in comparison to those of the individual polypeptides. The data are consistent with the EH heterodimer forming the peripheral stalk(s) in the A-ATPase in a fashion analogous to that of the related vacuolar ATPase. [Copyright &y& Elsevier]

The rotary bacterial flagellar motor is remarkable in biochemistry for its highly synchronized operation and amplification during switching of rotation sense. The motor is part of the flagellar basal body, a complex multi-protein assembly. Sensory and energy transduction depends on a core of six proteins that are adapted in different species to adjust torque and produce diverse switches. Motor response to chemotactic and environmental stimuli is driven by interactions of the core with small signal proteins. The initial protein interactions are propagated across a multi-subunit cytoplasmic ring to switch torque. Torque reversal triggers structural transitions in the flagellar filament to change motile behavior. Subtle variations in the core components invert or block switch operation. The mechanics of the flagellar switch have been studied with multiple approaches, from protein dynamics to single molecule and cell biophysics. The architecture, driven by recent advances in electron cryo-microscopy, is available for several species. Computational methods have correlated structure with genetic and biochemical databases. The design principles underlying the basis of switch ultra-sensitivity and its dependence on motor torque remain elusive, but tantalizing clues have emerged. This review aims to consolidate recent knowledge into a unified platform that can inspire new research strategies. [ABSTRACT FROM AUTHOR]

We present a chemomechanical network model of the rotary molecular motor F 1 -ATPase which quantitatively describes not only the rotary motor dynamics driven by ATP hydrolysis but also the ATP synthesis caused by forced reverse rotations. We observe a high reversibility of F 1 -ATPase, that is, the main cycle of ATP synthesis corresponds to the reversal of the main cycle in the hydrolysis-driven motor rotation. However, our quantitative analysis indicates that torque-induced mechanical slip without chemomechanical coupling occurs under high external torque and reduces the maximal efficiency of the free energy transduction to 40-80% below the optimal efficiency. Heat irreversibly dissipates not only through the viscous friction of the probe but also directly from the motor due to torque-induced mechanical slip. Such irreversible heat dissipation is a crucial limitation for achieving a 100% free-energy transduction efficiency with biological nanomachines because biomolecules are easily deformed by external torque.