Structure and Mechanisms of F-Type ATP Synthases.

F1Fo ATP synthases produce most of the ATP in the cell. F-type ATP synthases have been investigated for more than 50 years, but a full understanding of their molecular mechanisms has become possible only with the recent structures of complete, functionally competent complexes determined by electron cryo-microscopy (cryo-EM). High-resolution cryo-EM structures offer a wealth of unexpected new insights. The catalytic F1 head rotates with the central γ-subunit for the first part of each ATP-generating power stroke. Joint rotation is enabled by subunit δ/OSCP acting as a flexible hinge between F1 and the peripheral stalk. Subunit a conducts protons to and from the c-ring rotor through two conserved aqueous channels. The channels are separated by ∼6 Šin the hydrophobic core of Fo, resulting in a strong local field that generates torque to drive rotary catalysis in F1. The structure of the chloroplast F1Fo complex explains how ATPase activity is turned off at night by a redox switch. Structures of mitochondrial ATP synthase dimers indicate how they shape the inner membrane cristae. The new cryo-EM structures complete our picture of the ATP synthases and reveal the unique mechanism by which they transform an electrochemical membrane potential into biologically useful chemical energy.

Horizontal membrane-intrinsic α-helices in the stator a-subunit of an F-type ATP synthase.

ATP, the universal energy currency of cells, is produced by F-type ATP synthases, which are ancient, membrane-bound nanomachines. F-type ATP synthases use the energy of a transmembrane electrochemical gradient to generate ATP by rotary catalysis. Protons moving across the membrane drive a rotor ring composed of 8-15 c-subunits. A central stalk transmits the rotation of the c-ring to the catalytic F1 head, where a series of conformational changes results in ATP synthesis. A key unresolved question in this fundamental process is how protons pass through the membrane to drive ATP production. Mitochondrial ATP synthases form V-shaped homodimers in cristae membranes. Here we report the structure of a native and active mitochondrial ATP synthase dimer, determined by single-particle electron cryomicroscopy at 6.2 Å resolution. Our structure shows four long, horizontal membrane-intrinsic α-helices in the a-subunit, arranged in two hairpins at an angle of approximately 70° relative to the c-ring helices. It has been proposed that a strictly conserved membrane-embedded arginine in the a-subunit couples proton translocation to c-ring rotation. A fit of the conserved carboxy-terminal a-subunit sequence places the conserved arginine next to a proton-binding c-subunit glutamate. The map shows a slanting solvent-accessible channel that extends from the mitochondrial matrix to the conserved arginine. Another hydrophilic cavity on the lumenal membrane surface defines a direct route for the protons to an essential histidine-glutamate pair. Our results provide unique new insights into the structure and function of rotary ATP synthases and explain how ATP production is coupled to proton translocation.

Overexpression, purification, enzymatic and microscopic characterization of recombinant mycobacterial F-ATP synthase.

The F-ATP synthase is an essential enzyme in mycobacteria, including the pathogenic Mycobacterium tuberculosis. Several new compounds in the TB-drug pipeline target the F-ATP synthase. In light of the importance and pharmacological attractiveness of this novel antibiotic target, tools have to be developed to generate a recombinant mycobacterial F1FO ATP synthase to achieve atomic insight and mutants for mechanistic and regulatory understanding as well as structure-based drug design. Here, we report the first genetically engineered, purified and enzymatically active recombinant M. smegmatis F1FO ATP synthase. The projected 2D- and 3D structures of the recombinant enzyme derived from negatively stained electron micrographs are presented. Furthermore, the first 2D projections from cryo-electron images are revealed, paving the way for an atomic resolution structure determination.

Membrane insertion of F0 c subunit of F0F1 ATPase depends on glycolipozyme MPIase and is stimulated by YidC.

The F0 c subunit of F0F1 ATPase (F0-c) possesses two membrane-spanning stretches with N- and C-termini exposed to the periplasmic (extracellular) side of the cytoplasmic membrane of E. coli. Although F0-c insertion has been extensively analyzed in vitro by means of protease protection assaying, it is unclear whether such assays allow elucidation of the insertion process faithfully, since the membrane-protected fragment, an index of membrane insertion, is a full-length polypeptide of F0-c, which is the same as the protease-resistant conformation without membrane insertion. We found that the protease-resistant conformation could be discriminated from membrane-insertion by including octyl glucoside on protease digestion. By means of this system, we found that F0-c insertion depends on MPIase, a glycolipozyme involved in membrane insertion, and is stimulated by YidC. In addition, we found that acidic phospholipids PG and CL transform F0-c into a protease-resistant form, while MPIase prevents the acquisition of such a protease-resistant conformation.

Subnanometre-resolution structure of the intact Thermus thermophilus H+-driven ATP synthase.

Ion-translocating rotary ATPases serve either as ATP synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. During ATP synthesis, ion translocation through the membrane-bound region of the complex causes rotation of a central rotor that drives conformational changes and ATP synthesis in the catalytic region of the complex. There are no structural models available for the intact membrane region of any ion-translocating rotary ATPase. Here we present a 9.7 Å resolution map of the H(+)-driven ATP synthase from Thermus thermophilus obtained by electron cryomicroscopy of single particles in ice. The 600-kilodalton complex has an overall subunit composition of A(3)B(3)CDE(2)FG(2)IL(12). The membrane-bound motor consists of a ring of L subunits and the carboxy-terminal region of subunit I, which are equivalent to the c and a subunits of most other rotary ATPases, respectively. The map shows that the ring contains 12 L subunits and that the I subunit has eight transmembrane helices. The L(12) ring and I subunit have a surprisingly small contact area in the middle of the membrane, with helices from the I subunit making contacts with two different L subunits. The transmembrane helices of subunit I form bundles that could serve as half-channels across the membrane, with the first half-channel conducting protons from the periplasm to the L(12) ring and the second half-channel conducting protons from the L(12) ring to the cytoplasm. This structure therefore suggests the mechanism by which a transmembrane proton motive force is converted to rotation in rotary ATPases.

A unique mechanism of curcumin inhibition on F1 ATPase.

ATP synthase (F-ATPase) function depends upon catalytic and rotation cycles of the F1 sector. Previously, we found that F1 ATPase activity is inhibited by the dietary polyphenols, curcumin, quercetin, and piceatannol, but that the inhibitory kinetics of curcumin differs from that of the other two polyphenols (Sekiya et al., 2012, 2014). In the present study, we analyzed Escherichia coli F1 ATPase rotational catalysis to identify differences in the inhibitory mechanism of curcumin versus quercetin and piceatannol. These compounds did not affect the 120° rotation step for ATP binding and ADP release, though they significantly increased the catalytic dwell duration for ATP hydrolysis. Analysis of wild-type F1 and a mutant lacking part of the piceatannol binding site (γΔ277-286) indicates that curcumin binds to F1 differently from piceatannol and quercetin. The unique inhibitory mechanism of curcumin is also suggested from its effect on F1 mutants with defective ß-γ subunit interactions (γMet23 to Lys) or ß conformational changes (ßSer174 to Phe). These results confirm that smooth interaction between each ß subunit and entire γ subunit in F1 is pertinent for rotational catalysis.

Microscopy of single F(o) F(1) -ATP synthases--the unraveling of motors, gears, and controls.

Optical microscopy of single F(1) -ATPase and F(o) F(1) -ATP synthases started 15 years ago. Direct demonstration of ATP-driven subunit rotation by videomicroscopy became the new exciting tool to analyze the conformational changes of this enzyme during catalysis. Stimulated by these experiments, technical improvements for higher time resolution, better angular resolution, and reduced viscous drag were developed rapidly. Optics and single-molecule enzymology were entangled to benefit both biochemists and microscopists. Today, several single-molecule microscopy methods are established including controls for the precise nanomanipulation of individual enzymes in vitro. Förster resonance energy transfer, which has been used for simultaneous monitoring of conformational changes of different parts of this rotary motor, is one of them and may become the tool for the analysis of single F(o) F(1) -ATP synthases in membranes of living cells. Here, breakthrough experiments are critically reviewed and challenges are discussed for the future microscopy of single ATP synthesizing enzymes at work.

A high-throughput genetically directed protein crosslinking analysis reveals the physiological relevance of the ATP synthase 'inserted' state.

ATP synthase, a highly conserved protein complex that has a subunit composition of α3 ß3 γδεab2 c8-15 for the bacterial enzyme, is a key player in supplying energy to living organisms. This protein complex consists of a peripheral F1 sector (α3 ß3 γδε) and a membrane-integrated Fo sector (ab2 c8-15 ). Structural analyses of the isolated protein components revealed that, remarkably, the C-terminal domain of its ε-subunit seems to adopt two dramatically different structures, but the physiological relevance of this conformational change remains largely unknown. In an attempt to decipher this, we developed a high-throughput in vivo protein photo-cross-linking analysis pipeline based on the introduction of the unnatural amino acid into the target protein via the scarless genome-targeted site-directed mutagenesis technique, and probing the cross-linked products via the high-throughput polyacrylamide gel electrophoresis technique. Employing this pipeline, we examined the interactions involving the C-terminal helix of the ε-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the bacterial ATP synthase exists as an equilibrium between the 'inserted' and 'noninserted' state in cells, maintaining a moderate but significant level of net ATP synthesis when shifting to the former upon exposing to unfavorable energetically stressful conditions. Such a mechanism allows the bacterial ATP synthases to proportionally and instantly switch between two reversible functional states in responding to changing environmental conditions. Importantly, this high-throughput approach could allow us to decipher the physiological relevance of protein-protein interactions identified under in vitro conditions or to unveil novel physiological context-dependent protein-protein interactions that are unknown before.

Acidic lipids, H(+)-ATPases, and mechanism of oxidative phosphorylation. Physico-chemical ideas 30 years after P. Mitchell's Nobel Prize award.

Peter D. Mitchell, who was awarded the Nobel Prize in Chemistry 30 years ago, in 1978, formulated the chemiosmotic theory of oxidative phosphorylation. This review initially analyzes the major aspects of this theory, its unresolved problems, and its modifications. A new physico-chemical mechanism of energy transformation and coupling of oxidation and phosphorylation is then suggested based on recent concepts regarding proteins, including ATPases that work as molecular motors, and acidic lipids that act as hydrogen ion (H(+)) carriers. According to this proposed mechanism, the chemical energy of a redox substrate is transformed into nonequilibrium states of electron-transporting chain (ETC) coupling proteins. This leads to nonequilibrium pumping of H(+) into the membrane. An acidic lipid, cardiolipin, binds with this H(+) and carries it to the ATP-synthase along the membrane surface. This transport generates gradients of surface tension or electric field along the membrane surface. Hydrodynamic effects on a nanolevel lead to rotation of ATP-synthase and finally to the release of ATP into aqueous solution. This model also explains the generation of a transmembrane protonmotive force that is used for regulation of transmembrane transport, but is not necessary for the coupling of electron transport and ATP synthesis.

Ca2+ -dependent interaction of S100A1 with F1-ATPase leads to an increased ATP content in cardiomyocytes.

S100A1, a Ca(2+)-sensing protein of the EF-hand family that is expressed predominantly in cardiac muscle, plays a pivotal role in cardiac contractility in vitro and in vivo. It has recently been demonstrated that by restoring Ca(2+) homeostasis, S100A1 was able to rescue contractile dysfunction in failing rat hearts. Myocardial contractility is regulated not only by Ca(2+) homeostasis but also by energy metabolism, in particular the production of ATP. Here, we report a novel interaction of S100A1 with mitochondrial F(1)-ATPase, which affects F(1)-ATPase activity and cellular ATP production. In particular, cardiomyocytes that overexpress S100A1 exhibited a higher ATP content than control cells, whereas knockdown of S100A1 expression decreased ATP levels. In pull-down experiments, we identified the alpha- and beta-chain of F(1)-ATPase to interact with S100A1 in a Ca(2+)-dependent manner. The interaction was confirmed by colocalization studies of S100A1 and F(1)-ATPase and the analysis of the S100A1-F(1)-ATPase complex by gel filtration chromatography. The functional impact of this association is highlighted by an S100A1-mediated increase of F(1)-ATPase activity. Consistently, ATP synthase activity is reduced in cardiomyocytes from S100A1 knockout mice. Our data indicate that S100A1 might play a key role in cardiac energy metabolism.

The plasma membrane H+-ATPase is related to the development of salicylic acid-induced thermotolerance in pea leaves.

The plasma membrane H(+)-ATPase (PM H(+)-ATPase, EC.3.6.1.35) plays a key role in the plant response to environmental stress. In this study, a possible mechanistic link between the PM H(+)-ATPase and salicylic acid (SA)-induced thermotolerance was investigated in pea (Pisum sativum L. cv. NingXia) leaves. The burst of free SA in response to heat acclimation (38 +/- 0.5 degrees C) was observed, and peaks appeared subsequently both in activity and amount of PM H(+)-ATPase in pea leaves during heat acclimation. Similarly, exogenous SA also triggered the two peaks in the room temperature (25 +/- 0.5 degrees C). Paclobutrazol (PAC) was employed to infiltrate onto pea leaves prior to heat acclimation treatment. The results showed that the peaks of both free SA and activity of PM H(+)-ATPase still occurred after the PAC pretreatment. In acquired thermotolerance assessment (malondialdehyde content and degree of wilting), spraying SA and fusicoccin (FC, the activator of PM H(+)-ATPase) separately could protect pea leaves from heat injury. Results from RT-PCR and western blotting analysis indicated that the increase in activity of the PM H(+)-ATPase was due to its transcriptional and translational regulation. The subcellular localizations of PM H(+)-ATPase after the FC or SA pretreatment also showed that the PM H(+)-ATPase is important to maintain the integrity of plasma membrane against the heat stress. Taken together, these results suggest PM H(+)-ATPase is related to the development of SA-induced thermotolerance in pea leaves.

NMR solution structure of the N-terminal domain of subunit E (E1-52) of A1AO ATP synthase from Methanocaldococcus jannaschii.

The N-termini of E and H of A1AO ATP synthase have been shown to interact and an NMR structure of N-terminal H1-47 has been solved recently. In order to understand the E-H assembly and the N-terminal structure of E, the truncated construct E1-52 of Methanocaldococcus jannaschii A1AO ATP synthase was produced, purified and the solution structure of E1-52 was determined by NMR spectroscopy. The protein is 60.5 A in length and forms an alpha helix between the residues 8-48. The molecule is amphipathic with a strip of hydrophobic residues, discussed as a possible helix-helix interaction with neighboring subunit H.

Rotary protein motors.

Three protein motors have been unambiguously identified as rotary engines: the bacterial flagellar motor and the two motors that constitute ATP synthase (F(0)F(1) ATPase). Of these, the bacterial flagellar motor and F(0) motors derive their energy from a transmembrane ion-motive force, whereas the F(1) motor is driven by ATP hydrolysis. Here, we review the current understanding of how these protein motors convert their energy supply into a rotary torque.

Cryo-EM structure of the homohexameric T3SS ATPase-central stalk complex reveals rotary ATPase-like asymmetry.

Many Gram-negative bacteria, including causative agents of dysentery, plague, and typhoid fever, rely on a type III secretion system - a multi-membrane spanning syringe-like apparatus - for their pathogenicity. The cytosolic ATPase complex of this injectisome is proposed to play an important role in energizing secretion events and substrate recognition. We present the 3.3 Å resolution cryo-EM structure of the enteropathogenic Escherichia coli ATPase EscN in complex with its central stalk EscO. The structure shows an asymmetric pore with different functional states captured in its six catalytic sites, details directly supporting a rotary catalytic mechanism analogous to that of the heterohexameric F1/V1-ATPases despite its homohexameric nature. Situated at the C-terminal opening of the EscN pore is one molecule of EscO, with primary interaction mediated through an electrostatic interface. The EscN-EscO structure provides significant atomic insights into how the ATPase contributes to type III secretion, including torque generation and binding of chaperone/substrate complexes.

Structure of a bacterial ATP synthase.

ATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed the Bacillus PS3 ATP synthase in Eschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunit ε shows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

Temperature dependence of the rotation and hydrolysis activities of F1-ATPase.

F(1)-ATPase, a water-soluble portion of the enzyme ATP synthase, is a rotary molecular motor driven by ATP hydrolysis. To learn how the kinetics of rotation are regulated, we have investigated the rotational characteristics of a thermophilic F(1)-ATPase over the temperature range 4-50 degrees C by attaching a polystyrene bead (or bead duplex) to the rotor subunit and observing its rotation under a microscope. The apparent rate of ATP binding estimated at low ATP concentrations increased from 1.2 x 10(6) M(-1) s(-1) at 4 degrees C to 4.3 x 10(7) M(-1) s(-1) at 40 degrees C, whereas the torque estimated at 2 mM ATP remained around 40 pN.nm over 4-50 degrees C. The rotation was stepwise at 4 degrees C, even at the saturating ATP concentration of 2 mM, indicating the presence of a hitherto unresolved rate-limiting reaction that occurs at ATP-waiting angles. We also measured the ATP hydrolysis activity in bulk solution at 4-65 degrees C. F(1)-ATPase tends to be inactivated by binding ADP tightly. Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30 degrees C, equilibrium between the active and inactive forms was reached within 2 s, the majority being inactive. Rapid inactivation at high temperatures is consistent with the physiological role of this enzyme, ATP synthesis, in the thermophile.

Structure of the cytosolic part of the subunit b-dimer of Escherichia coli F0F1-ATP synthase.

The structure of the external stalk and its function in the catalytic mechanism of the F(0)F(1)-ATP synthase remains one of the important questions in bioenergetics. The external stalk has been proposed to be either a rigid stator that binds F(1) or an elastic structural element that transmits energy from the small rotational steps of subunits c to the F(1) sector during catalysis. We employed proteomics, sequence-based structure prediction, molecular modeling, and electron spin resonance spectroscopy using site-directed spin labeling to understand the structure and interfacial packing of the Escherichia coli b-subunit homodimer external stalk. Comparisons of bacterial, cyanobacterial, and plant b-subunits demonstrated little sequence similarity. Supersecondary structure predictions, however, show that all compared b-sequences have extensive heptad repeats, suggesting that the proteins all are capable of packing as left-handed coiled-coils. Molecular modeling subsequently indicated that b(2) from the E. coli ATP synthase could pack into stable left-handed coiled-coils. Thirty-eight substitutions to cysteine in soluble b-constructs allowed the introduction of spin labels and the determination of intersubunit distances by ESR. These distances correlated well with molecular modeling results and strongly suggest that the E. coli subunit b-dimer can stably exist as a left-handed coiled-coil.

Neither helix in the coiled coil region of the axle of F1-ATPase plays a significant role in torque production.

F(1)-ATPase is an ATP-driven rotary molecular motor in which the central gamma-subunit rotates inside the cylinder made of alpha(3)beta(3) subunits. The amino and carboxy termini of the gamma-subunit form the axle, an alpha-helical coiled coil that deeply penetrates the stator cylinder. We previously truncated the axle step by step, starting with the longer carboxy terminus and then cutting both termini at the same levels, resulting in a slower yet considerably powerful rotation. Here we examine the role of each helix by truncating only the carboxy terminus by 25-40 amino-acid residues. Longer truncation impaired the stability of the motor complex severely: 40 deletions failed to yield rotating the complex. Up to 36 deletions, however, the mutants produced an apparent torque at nearly half of the wild-type torque, independent of truncation length. Time-averaged rotary speeds were low because of load-dependent stumbling at 120 degrees intervals, even with saturating ATP. Comparison with our previous work indicates that half the normal torque is produced at the orifice of the stator. The very tip of the carboxy terminus adds the other half, whereas neither helix in the middle of the axle contributes much to torque generation and the rapid progress of catalysis. None of the residues of the entire axle played a specific decisive role in rotation.

Fluid mechanical matching of H+-ATP synthase subunit c-ring with lipid membranes revealed by 2H solid-state NMR.

The F(1)F(o)-ATP synthase utilizes the transmembrane H(+) gradient for the synthesis of ATP. F(o) subunit c-ring plays a key role in transporting H(+) through F(o) in the membrane. We investigated the interactions of Escherichia coli subunit c with dimyristoylphosphatidylcholine (DMPC-d(54)) at lipid/protein ratios of 50:1 and 20:1 by means of (2)H-solid-state NMR. In the liquid-crystalline state of DMPC, the (2)H-NMR moment values and the order parameter (S(CD)) profile were little affected by the presence of subunit c, suggesting that the bilayer thickness in the liquid-crystalline state is matched to the transmembrane hydrophobic surface of subunit c. On the other hand, hydrophobic mismatch of subunit c with the lipid bilayer was observed in the gel state of DMPC. Moreover, the viscoelasticity represented by a square-law function of the (2)H-NMR relaxation was also little influenced by subunit c in the fluid phase, in contrast with flexible nonionic detergents or rigid additives. Thus, the hydrophobic matching of the lipid bilayer to subunit c involves at least two factors, the hydrophobic length and the fluid mechanical property. These findings may be important for the torque generation in the rotary catalytic mechanism of the F(1)F(o)-ATPse molecular motor.

Functional halt positions of rotary FOF1-ATPase correlated with crystal structures.

The F(O)F(1)-ATPase is a rotary molecular motor. Driven by ATP-hydrolysis, its central shaft rotates in 80 degrees and 40 degrees steps, interrupted by catalytic and ATP-waiting dwells. We recorded rotations and halts by means of microvideography in laboratory coordinates. A correlation with molecular coordinates was established by using an engineered pair of cysteines that, under oxidizing conditions, formed zero-length cross-links between the rotor and the stator in an orientation as found in crystals. The fixed orientation coincided with that of the catalytic dwell, whereas the ATP waiting dwell was displaced from it by +40 degrees . In crystals, the convex side of the cranked central shaft faces an empty nucleotide binding site, as if holding it open for arriving ATP. Functional studies suggest that three sites are occupied during a catalytic dwell. Our data imply that the convex side faces a nucleotide-occupied rather than an empty site. The enzyme conformation in crystals seems to differ from the conformation during either dwell of the active enzyme. A revision of current schemes of the mechanism is proposed.