Functional incorporation of chimeric b subunits into F1Fo ATP synthase

F(1)F(o) ATP synthases function by a rotary mechanism. The enzyme's peripheral stalk serves as the stator that holds the F(1) sector and its catalytic sites against the movement of the rotor. In Escherichia coli, the peripheral stalk is a homodimer of identical b subunits, but photosynthetic bacteria have open reading frames for two different b-like subunits thought to form heterodimeric b/b' peripheral stalks. Chimeric b subunit genes have been constructed by substituting sequence from the Thermosynechococcus elongatus b and b' genes in the E. coli uncF gene, encoding the b subunit. The recombinant genes were expressed alone and in combination in the E. coli deletion strain KM2 (Deltab). Although not all of the chimeric subunits were incorporated into F(1)F(o) ATP synthase complexes, plasmids expressing either chimeric b(E39-I86) or b'(E39-I86) were capable of functionally complementing strain KM2 (Deltab). Strains expressing these subunits grew better than cells with smaller chimeric segments, such as those expressing the b'(E39-D53) or b(L54-I86) subunit, indicating intragenic suppression. In general, the chimeric subunits modeled on the T. elongatus b subunit proved to be more stable than the b' subunit in vitro. Coexpression of the b(E39-I86) and b'(E39-I86) subunits in strain KM2 (Deltab) yielded F(1)F(o) complexes containing heterodimeric peripheral stalks composed of both subunits.     

F-ATPase: forced full rotation of the rotor despite covalent cross-link with the stator

In ATP synthase (F(O)F(1)-ATPase) ion flow through the membrane-intrinsic portion, F(O), drives the central "rotor", subunits c(10)epsilongamma, relative to the "stator" ab(2)delta(alphabeta)(3). This converts ADP and P(i) into ATP. Vice versa, ATP hydrolysis drives the rotation backwards. Covalent cross-links between rotor and stator subunits have been shown to inhibit these activities. Aiming at the rotary compliance of subunit gamma we introduced disulfide bridges between gamma (rotor) and alpha or beta (stator). We engineered cysteine residues into positions located roughly at the "top," "center," and "bottom" parts of the coiled-coil portion of gamma and suitable residues on alpha or beta. This part of gamma is located at the center of the (alphabeta)(3) domain with its C-terminal part at the top of F(1) and the bottom part close to the F(O) complex. Disulfide bridge formation under oxidizing conditions was quantitative as shown by SDS-polyacrylamide gel electrophoresis and immunoblotting. As expected both the ATPase activities and the yield of rotating subunits gamma dropped to zero when the cross-link was formed at the center (gammaL262C <--> alphaA334C) and bottom (gammaCys(87) <--> betaD380C) positions. But much to our surprise disulfide bridging impaired neither ATP hydrolysis activity nor the full rotation of gamma and the enzyme-generated torque of oxidized F(1), which had been engineered at the top position (gammaA285C <--> alphaP280C). Apparently the high torque of this rotary engine uncoiled the alpha-helix and forced amino acids at the C-terminal portion of gamma into full rotation around their dihedral (Ramachandran) angles. This conclusion was supported by molecular dynamics simulations: If gammaCys(285)-Val(286) are attached covalently to (alphabeta)(3) and gammaAla(1)-Ser(281) is forced to rotate, gammaGly(282)-Ala(284) can serve as cardan shaft.     

Reverse engineering a protein: the mechanochemistry of ATP synthase

ATP synthase comprises two rotary motors in one. The F(1) motor can generate a mechanical torque using the hydrolysis energy of ATP. The F(o) motor generates a rotary torque in the opposite direction, but it employs a transmembrane proton motive force. Each motor can be reversed: The F(o) motor can drive the F(1) motor in reverse to synthesize ATP, and the F(1) motor can drive the F(o) motor in reverse to pump protons. Thus ATP synthase exhibits two of the major energy transduction pathways employed by the cell to convert chemical energy into mechanical force. Here we show how a physical analysis of the F(1) and F(o) motors can provide a unified view of the mechanochemical principles underlying these energy transducers.     

Inter-subunit rotation and elastic power transmission in F0F1-ATPase

ATP synthase (F-ATPase) produces ATP at the expense of ion-motive force or vice versa. It is composed from two motor/generators, the ATPase (F1) and the ion translocator (F0), which both are rotary steppers. They are mechanically coupled by 360 degrees rotary motion of subunits against each other. The rotor, subunits gamma(epsilon)C10-14, moves against the stator, (alphabeta)3delta(ab2). The enzyme copes with symmetry mismatch (C3 versus C10-14) between its two motors, and it operates robustly in chimeric constructs or with drastically modified subunits. We scrutinized whether an elastic power transmission accounts for these properties. We used the curvature of fluorescent actin filaments, attached to the rotating c ring, as a spring balance (flexural rigidity of 8.10(-26) N x m2) to gauge the angular profile of the output torque at F0 during ATP hydrolysis by F1. The large average output torque (56 pN nm) proved the absence of any slip. Angular variations of the torque were small, so that the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the three-fold stepping and high activation barrier (>40 kJ/mol) of the driving motor (F1) itself, the rather constant output torque seen by F0 implied a soft elastic power transmission between F1 and F0. It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate under load of the two counteracting and stepping motors/generators.     

The C-H Peripheral Stalk Base: A Novel Component in V1-ATPase Assembly

Vacuolar ATPases (V-ATPases) are molecular machines responsible for creating electrochemical gradients and preserving pH-dependent cellular compartments by way of proton translocation across the membrane. V-ATPases employ a dynamic rotary mechanism that is driven by ATP hydrolysis and the central rotor stalk. Regulation of this rotational catalysis is the result of a reversible V₁V_o-domain dissociation that is required to preserve ATP during instances of cellular starvation. Recently the method by which the free V₁-ATPase abrogates the hydrolytic breakdown of ATP upon dissociating from the membrane has become increasingly clear. In this instance the central stalk subunit F adopts an extended conformation to engage in a bridging interaction tethering the rotor and stator components together. However, the architecture by which this mechanism is stabilized has remained ambiguous despite previous work. In an effort to elucidate the method by which the rotational catalysis is maintained, the architecture of the peripheral stalks and their respective binding interactions was investigated using cryo-electron microscopy. In addition to confirming the bridging interaction exuded by subunit F for the first time in a eukaryotic V-ATPase, subunits C and H are seen interacting with one another in a tight interaction that provides a base for the three EG peripheral stalks. The formation of a CE₃G₃H sub-assembly appears to be unique to the dissociated V-ATPase and highlights the stator architecture in addition to revealing a possible intermediate in the assembly mechanism of the free V₁-ATPase.     

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.     

Subunit H of the vacuolar (H+) ATPase inhibits ATP hydrolysis by the free V1 domain by interaction with the rotary subunit F

The vacuolar (H+) ATPases (V-ATPases) are large, multimeric proton pumps that, like the related family of F1F0 ATP synthases, employ a rotary mechanism. ATP hydrolysis by the peripheral V1 domain drives rotation of a rotary complex (the rotor) relative to the stationary part of the enzyme (the stator), leading to proton translocation through the integral V0 domain. One mechanism of regulating V-ATPase activity in vivo involves reversible dissociation of the V1 and V0 domains. Unlike the corresponding domains in F1F0, the dissociated V1 domain does not hydrolyze ATP, and the free V0 domain does not passively conduct protons. These properties are important to avoid generation of an uncoupled ATPase activity or an unregulated proton conductance upon dissociation of the complex in vivo. Previous results (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767) showed that subunit H (part of the stator) inhibits ATP hydrolysis by free V1. To test the hypothesis that subunit H accomplishes this by bridging rotor and stator in free V1, cysteine-mediated cross-linking studies were performed. Unique cysteine residues were introduced over the surface of subunit H from yeast by site-directed mutagenesis and used as the site of attachment of the photo-activated cross-linking reagent maleimido benzophenone. After UV-activated cross-linking, cross-linked products were identified by Western blot using subunit-specific antibodies. The results indicate that the subunit H mutant S381C shows cross-linking between subunit H and subunit F (a rotor subunit) in the free V1 domain but not in the intact V1V0 complex. These results indicate that subunits H and F are proximal in free V1, supporting the hypothesis that subunit H inhibits free V1 by bridging the rotary and stator domains.     

Dynamic and coordinating domain motions in the active subunits of the F1-ATPase molecular motor

F1-ATPase is a rotary molecular motor crucial for various cellular functions. In F1-ATPase, the rotation of the gammadeltaepsilon subunits against the hexameric alpha(3)beta(3) subunits is highly coordinative, driven by ATP hydrolysis and structural changes at three beta subunits. However, the dynamical and coordinating structural transitions in the beta subunits are not fully understood at the molecular level. Here we examine structural transitions and domain motions in the active subunits of F1-ATPase via dynamical domain analysis of the alpha(3)beta(3)gammadeltaepsilon complex. The domain movement and hinge axes and bending residues have been identified and determined for various conformational changes of the beta-subunits. P-loop and the ATP-binding pocket are for the first time found to play essential mechanical functions additional to the catalytic roles. The cooperative conformational changes pertaining to the rotary mechanism of F1-ATPase appears to be more complex than Boyer's 'bi-site' activity. These findings provide unique molecular insights into dynamic and cooperative domain motions in F1-ATPase.     

Characterization of the flexibility of the peripheral stalk of prokaryotic rotary A‐ATPases by atomistic simulations

Rotary ATPases are involved in numerous physiological processes, with the three distinct types (F/A/V‐ATPases) sharing functional properties and structural features. The basic mechanism involves the counter rotation of two motors, a soluble ATP hydrolyzing/synthesizing domain and a membrane‐embedded ion pump connected through a central rotor axle and a stator complex. Within the A/V‐ATPase family conformational flexibility of the EG stators has been shown to accommodate catalytic cycling and is considered to be important to function. For the A‐ATPase three EG structures have been reported, thought to represent conformational states of the stator during different stages of rotary catalysis. Here we use long, detailed atomistic simulations to show that those structures are conformers explored through thermal fluctuations, but do not represent highly populated states of the EG stator in solution. We show that the coiled coil tail domain has a high persistence length (～100 nm), but retains the ability to adapt to different conformational states through the presence of two hinge regions. Moreover, the stator network of the related V‐ATPase has been suggested to adapt to subunit interactions in the collar region in addition to the nucleotide occupancy of the catalytic domain. The MD simulations reported here, reinforce this observation showing that the EG stators have enough flexibility to adapt to significantly different structural re‐arrangements and accommodate structural changes in the catalytic domain whilst resisting the large torque generated by catalytic cycling. These results are important to understand the role the stators play in the rotary‐ATPase mechanism. Proteins 2016; 84:1203–1212. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

A structure-based model for the synthesis and hydrolysis of ATP by F1-ATPase

Many essential functions of living cells are performed by nanoscale protein motors. The best characterized of these is F(o)F1-ATP synthase, the smallest rotary motor. This rotary motor catalyzes the synthesis of ATP with high efficiency under conditions where the reactants (ADP, H2PO4(-)) and the product (ATP) are present in the cell at similar concentrations. We present a detailed structure-based kinetic model for the mechanism of action of F1-ATPase and demonstrate the role of different protein conformations for substrate binding during ATP synthesis and ATP hydrolysis. The model shows that the pathway for ATP hydrolysis is not simply the pathway for ATP synthesis in reverse. The findings of the model also explain why the cellular concentration of ATP does not inhibit ATP synthesis.     

Both rotor and stator subunits are necessary for efficient binding of F1 to F0 in functionally assembled Escherichia coli ATP synthase

In F1F0-ATP synthase, the subunit b2delta complex comprises the peripheral stator bound to subunit a in F0 and to the alpha3beta3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F0 subunits to the binding of F1 within the assembled holoenzyme, the isolated subunit b dimer, ab2 subcomplex, and fully assembled F0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F1 (at gammaCys106 and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F1 dissociation constants of 2.7 nm for the binding of F0 and 9-10 nm for both the ab2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F1 to F0, whereas subunit a does not contribute significantly.     

Second stalk of ATP synthase. Cross-linking of gamma subunit in F1 to truncated Fob subunit prevents ATP hydrolysis

ATP synthase consists of two portions, F(1) and F(o), connected by two stalks: a central rotor stalk containing gamma and epsilon subunits and a peripheral, second stalk formed by delta and two copies of F(o)b subunits. The second stalk is expected to keep the stator subunits from spinning along with the rotor. We isolated a TF(1)-b'(2) complex (alpha(3)beta(3)gammadeltaepsilonb'(2)) of a thermophilic Bacillus PS3, in which b' was a truncated cytoplasmic fragment of F(o)b subunit, and introduced a cysteine at its N terminus (bc'). Association of b'(2) or bc'(2) with TF(1) did not have significant effect on ATPase activity. A disulfide bond between the introduced cysteine of bc' and cysteine 109 of gamma subunit was readily formed, and this cross-link caused inactivation of ATPase. This implies that F(o)b subunit bound to stator subunits of F(1) with enough strength to resist rotation of gamma subunit and to prevent catalysis. Contrary to this apparent tight binding, some detergents such as lauryldodecylamine oxide tend to cause release of b'(2) from TF(1).     

The Na(+)-translocating F(1)F(0) ATP synthase of Propionigenium modestum: mechanochemical insights into the F(0) motor that drives ATP synthesis

The ATP synthase of Propionigenium modestum encloses a rotary motor involved in the production of ATP from ADP and inorganic phosphate utilizing the free energy of an electrochemical Na(+) ion gradient. This enzyme clearly belongs to the family of F(1)F(0) ATP synthases and uses exclusively Na(+) ions as the physiological coupling ion. The motor domain, F(0), comprises subunit a and the b subunit dimer which are part of the stator and the subunit c oligomer acting as part of the rotor. During ATP synthesis, Na(+) translocation through F(0) proceeds from the periplasm via the stator channel (subunit a) onto a Na(+) binding site of the rotor (subunit c). Upon rotation of the subunit c oligomer versus subunit a, the occupied rotor site leaves the interface with the stator and the Na(+) ion can freely dissociate into the cytoplasm. Recent experiments demonstrate that the membrane potential is crucial for ATP synthesis under physiological conditions. These findings support the view that voltage generates torque in F(0), which drives the rotation of the gamma subunit thus liberating tightly bound ATP from the catalytic sites in F(1). We suggest a mechanochemical model for the transduction of transmembrane Na(+)-motive force into rotary torque by the F(0) motor that can account quantitatively for the experimental data.     

Mechanism of the F(1)F(0)-type ATP synthase, a biological rotary motor

The F(1)F(0)-type ATP synthase is a key enzyme in cellular energy interconversion. During ATP synthesis, this large protein complex uses a proton gradient and the associated membrane potential to synthesize ATP. It can also reverse and hydrolyze ATP to generate a proton gradient. The structure of this enzyme in different functional forms is now being rapidly elucidated. The emerging consensus is that the enzyme is constructed as two rotary motors, one in the F(1) part that links catalytic site events with movements of an internal rotor, and the other in the F(0) part, linking proton translocation to movements of this F(0) rotor. Although both motors can work separately, they must be connected together to interconvert energy. Evidence for the function of the rotary motor, from structural, genetic and biophysical studies, is reviewed here, and some uncertainties and remaining mysteries of the enzyme mechanism are also discussed.     

Cooperativity in the motor activities of the ATP-fueled molecular motors

Kinesin, myosin and F1-ATPase are multi-domain molecular motors with multiple catalytic subunits. The motor mechanochemics are achieved via the conversion of ATP hydrolysis energy into forces and motions. We find that the catalysis of these molecular motors do not follow the simple Michaelis-Menten mechanism. The motor activities, such as the hydrolysis or processive rates, of kinesin, myosin and F1-ATPase have a complex ATP-dependent cooperativity. To understand this complexity in kinetics and mechanochemics, we develop a conformation correlation theory of cooperativity for the ATP-fueled motor proteins. The quantitative analysis and simulations indicate that cooperativity is induced by the conformational coupling of binding states of different subunits and prevails in the motor activities.     

Rotation and structure of FoF1-ATP synthase

F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background.     

The rotor tip inside a bearing of a thermophilic F1-ATPase is dispensable for torque generation

F(1)-ATPase is an ATP-driven rotary molecular motor in which the central gamma-subunit rotates inside a stator cylinder made of alpha(3)beta(3) subunits. To elucidate the role of rotor-stator interactions in torque generation, we truncated the gamma-subunit at its carboxyl terminus, which forms an alpha helix that penetrates deeply into the stator cylinder. We used an alpha(3)beta(3)gamma subcomplex of F(1)-ATPase derived from thermophilic Bacillus PS3 and expressed it in Escherichia coli. We could obtain purified subcomplexes in which 14, 17, or 21 amino-acid residues were deleted. The rotary characteristics of the truncated mutants, monitored by attaching a duplex of 0.49-microm beads to the gamma-subunit, did not differ greatly from those of the wild-type over the ATP concentrations of 20 nM-2 mM, the most conspicuous effect being approximately 50% reduction in torque and approximately 70% reduction in the rate of ATP binding upon deletion of 21 residues. The ATP hydrolysis activity estimated in bulk samples was more seriously affected. The 21-deletion mutant, in particular, was >10-fold less active, but this is likely due to instability of this subcomplex. For torque generation, though not for rapid catalysis, most of the rotor-stator contacts on the deeper half of the penetrating portion of the gamma-subunit are dispensable.     

The rotary binding change mechanism of ATP synthases

The F(0)F(1) ATP synthase functions as a rotary motor where subunit rotation driven by a current of protons flowing through F(0) drives the binding changes in F(1) that are required for net ATP synthesis. Recent work that has led to the identification of components of the rotor and stator is reviewed. In addition, a model is proposed to describe the transmission of energy from four proton transport steps to the synthesis of one ATP. Finally, some of the requirements for efficient energy coupling by a rotary binding change mechanism are considered.     

Rotation of the c subunit oligomer in EF(0)EF(1) mutant cD61N

ATP synthases (F(0)F(1)-ATPases) mechanically couple ion flow through the membrane-intrinsic portion, F(0), to ATP synthesis within the peripheral portion, F(1). The coupling most probably occurs through the rotation of a central rotor (subunits c(10)epsilon gamma) relative to the stator (subunits ab(2)delta(alpha beta)(3)). The translocation of protons is conceived to involve the rotation of the ring of c subunits (the c oligomer) containing the essential acidic residue cD61 against subunits ab(2). In line with this notion, the mutants cD61N and cD61G have been previously reported to lack proton translocation. However, it has been surprising that the membrane-bound mutated holoenzyme hydrolyzed ATP but without translocating protons. Using detergent-solubilized and immobilized EF(0)F(1) and by application of the microvideographic assay for rotation, we found that the c oligomer, which carried a fluorescent actin filament, rotates in the presence of ATP in the mutant cD61N just as in the wild type enzyme. This observation excluded slippage among subunit gamma, the central rotary shaft, and the c oligomer and suggested free rotation without proton pumping between the oligomer and subunit a in the membrane-bound enzyme.