Search

How protons power rotation Synthesis of adenosine triphosphate (ATP) in mitochondria is accomplished by a large molecular machine, the F 1 F O ATP synthase. Proton translocation across the F O region that spans the mitochondrial inner membrane drives ATP synthesis in the F 1 region through a rotational mechanism. Guo et al. present a high-resolution structure of the dimeric F O complex from Saccharomyces cerevisiae , determined by electron microscopy. The structure gives insights into how proton translocation powers rotation and suggests how F O dimers bend the membrane to give mitochondria their characteristic cristae. Science , this issue p. 936

Fo·F1H+-ATPase/synthase in coupled plasma membrane vesicles of Paracoccus denitrificans catalyzes ATP hydrolysis and/or ATP synthesis with comparable enzyme turnover. Significant difference in pH-profile of these alternative activities is seen: decreasing pH from 8.0 to 7.0 results in reversible inhibition of hydrolytic activity, whereas ATP synthesis activity is not changed. The inhibition of ATPase activity upon acidification results from neither change in ADP(Mg2+)-induced deactivation nor the energy-dependent enzyme activation. Vmax, not apparent KmATP is affected by lowering the pH. Venturicidin noncompetitively inhibits ATP synthesis and coupled ATP hydrolysis, showing significant difference in the affinity to its inhibitory site depending on the direction of the catalysis. This difference cannot be attributed to variations of the substrate-enzyme intermediates for steady-state forward and back reactions or to possible equilibrium between ATP hydrolase and ATP synthase Fo·F1 modes of the opposite directions of catalysis. The data are interpreted as to suggest that distinct non-equilibrated molecular isoforms of Fo·F1 ATP synthase and ATP hydrolase exist in coupled energy-transducing membranes.

Cytochrome cbb3 (or C-type) oxidases are a highly divergent group and the least studied members of the heme-copper oxidases (HCOs) superfamily. HCOs couple the reduction of oxygen at the end of the respiratory chain to the active proton translocation across the membrane, contributing to establishment of an electrochemical gradient essential for ATP synthesis. Cbb3 oxidases exhibit unique structural and functional features and have an essential role in the metabolism of many clinically relevant human pathogens. Such characteristics make them a promising therapeutic target. Three subunits, N, O and P, comprise the core cbb3 complex, with N, the catalytic subunit, being highly conserved among all members of the HCO superfamily, including the A-type (aa3, mitochondrial-like) oxidases. An additional fourth subunit containing a single transmembrane (TM) helix was present in the first crystal structure of cbb3. This TM segment was recently proposed to be part of a novel protein CcoM, which was shown to have a putative role in the complex stability and assembly. In this work, we performed large-scale all-atom molecular dynamics simulations of the CcoNOPM complex to further characterize the interactions between subunit M and the core subunits and to determine whether the presence of the fourth subunit influences the water/proton channels previously described for the core complex. The previously proposed putative CcoNOPH complex is also assessed, and the potential functional redundancy of CcoM and CcoQ is discussed.

Mitochondrial F1FO-ATP synthase of the chlorophycean algae Polytomella sp. can be isolated as a highly stable dimeric complex of 1600kDa. It is composed of eight highly conserved orthodox subunits (α, β, γ, δ, e, OSCP, a, and c) and nine subunits (Asa1-9) that are exclusive of chlorophycean algae. The Asa subunits replace those that build up the peripheral stalk and the dimerization domains of the ATP synthase in other organisms. Little is known about the disposition of subunits Asa6, Asa8 and Asa9, that are predicted to have transmembrane stretches and that along with subunit a and a ring of c-subunits, seem to constitute the membrane-embedded Fo domain of the algal ATP synthase. Here, we over-expressed and purified the three Asa hydrophobic subunits and explored their interactions in vitro using a combination of immunochemical techniques, affinity chromatography, and an in vivo yeast-two hybrid assays. The results obtained suggest the following interactions Asa6-Asa6, Asa6-Asa8, Asa6-Asa9, Asa8-Asa8 and Asa8-Asa9. Cross-linking experiments carried out with the intact enzyme corroborated some of these interactions. Based on these results, we propose a model of the disposition of these hydrophobic subunits in the membrane-embedded sector of the algal ATP synthase. We also propose based on sequence analysis and hydrophobicity plots, that the algal subunit a is atypical in as much it lacks the first transmembrane stretch, exhibiting only four hydrophobic, tilted alpha helices.

Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Here we studied the mitochondrial F1FO-ATP synthase (or Complex V) from the photosynthetic eukaryote E. gracilis in detail. The enzyme was purified by a two-step chromatographic procedure and its subunit composition was resolved by a three-dimensional gel electrophoresis (BN/SDS/SDS). Twenty-two different subunits were identified by mass-spectrometry analyses among which the canonical α, β, γ, δ, ε, and OSCP subunits, and at least seven subunits previously found in Trypanosoma. The ADP/ATP carrier was also associated to the ATP synthase into a dimeric ATP synthasome. Single-particle analysis by transmission electron microscopy of the dimeric ATP synthase indicated that the structures of both the catalytic and central rotor parts are conserved while other structural features are original. These new features include a large membrane-spanning region joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring, the latter having not been reported for any mitochondrial F-ATPase.

In archaea the A1AO ATP synthase uses a transmembrane electrochemical potential to generate ATP, while the soluble A1 domain (subunits A3B3DF) alone can hydrolyse ATP. The three nucleotide-binding AB pairs form a barrel-like structure with a central orifice that hosts the rotating central stalk subunits DF. ATP binding, hydrolysis and product release cause a conformational change inside the A:B-interface, which enforces the rotation of subunits DF. Recently, we reported that subunit F is a stimulator of ATPase activity. Here, we investigated the nucleotide-dependent conformational changes of subunit F relative to subunit D during ATP hydrolysis in the A3B3DF complex of the Methanosarcina mazei Gö1 A-ATP synthase using single-molecule Förster resonance energy transfer. We found two conformations for subunit F during ATP hydrolysis. NMRC (Natl Medical Research Council, S’pore) Accepted version

Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6-20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b.

The Fo-a subunit of the Na+-transporting FoF1 ATP synthase from Propionigenium modestum plays a key role in Na+ transport. It forms half channels that allow Na+ to enter and leave the buried carboxyl group on Fo-c subunits. The essential Arg residue R226, which faces the carboxyl group of Fo-c subunits in the middle of transmembrane helix 5 of the Fo-a subunit, separates the cytoplasmic side and periplasmic half-channels. To elucidate contributions of other amino acid residues of transmembrane helix 5 using hybrid FoF1 (Fo from P. modestum and F1 from thermophilic Bacillus PS3), 25 residues were individually mutated to Cys, and effects of modification with the SH-modifying agent N-ethylmaleimide (NEM) on ATP synthesis and hydrolysis activity were analyzed. NEM significantly inhibited ATP synthesis and hydrolysis as well as proton pumping activities of A214C, G215C, A218C, I223C (cytoplasmic side from R226), and N230C (periplasmic side from R226) mutants and inhibited ATP synthesis activity of the K219C mutant (cytoplasmic side from R226). Thus, these residues contribute to the integrity of the Na+ half channel, and both half channels are present in the Fo-a subunit.

The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a "down" state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an "up" state, where the α-helices, devoid of ATP, enter the α3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme's hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual "open" conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis.

Thylakoid membrane-localized chloroplast ATP synthases use the proton motive force generated by photosynthetic electron transport to produce ATP from ADP. Although it is well known that the chloroplast ATP synthase is composed of more than 20 proteins with α3β3γ1ε1δ1I1II1III14IV1 stoichiometry, its biogenesis process is currently unclear. To unravel the molecular mechanisms underlying the biogenesis of chloroplast ATP synthase, we performed extensive screening for isolating ATP synthase mutants in Arabidopsis (Arabidopsis thaliana). In the recently identified bfa3 (biogenesis factors required for ATP synthase 3) mutant, the levels of chloroplast ATP synthase subunits were reduced to approximately 25% of wild-type levels. In vivo labeling analysis showed that assembly of the CF1 component of chloroplast ATP synthase was less efficient in bfa3 than in the wild type, indicating that BFA3 is required for CF1 assembly. BFA3 encodes a chloroplast stromal protein that is conserved in higher plants, green algae, and a few species of other eukaryotic algae, and specifically interacts with the CF1β subunit. The BFA3 binding site was mapped to a region in the catalytic site of CF1β. Several residues highly conserved in eukaryotic CF1β are crucial for the BFA3–CF1β interaction, suggesting a coevolutionary relationship between BFA3 and CF1β. BFA3 appears to function as a molecular chaperone that transiently associates with unassembled CF1β at its catalytic site and facilitates subsequent association with CF1α during assembly of the CF1 subcomplex of chloroplast ATP synthase.

F-type ATP synthases are reversible machinery that not only synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane, but also can hydrolyze ATP to pump ions under certain conditions. To prevent wasteful ATP hydrolysis, subunit ε in bacterial ATP synthases changes its conformation from the non-inhibitory down- to the inhibitory up-state at a low cellular ATP concentration. Recently, a crystal structure of the ε subunit in complex with ATP was solved in a non-biologically relevant dimeric form. Here, to derive the functional ATP binding site motif, we carried out molecular dynamics simulations and free energy calculations. Our results suggest that the ATP binding site markedly differs from the experimental resolved one; we observe a reorientation of several residues, which bind to ATP in the crystal structure. In addition we find that an Mg(2+) ion is coordinated by ATP, replacing interactions of the second chain in the crystal structure. Thus we demonstrate more generally the influence of crystallization effects on ligand binding sites and their respective binding modes. Furthermore, we propose a role for two highly conserved residues to control the ATP binding/unbinding event, which have not been considered before. Additionally our results provide the basis for the rational development of new biosensors based on subunit ε, as shown previously for novel sensors measuring the ATP concentration in cells.

Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases.

A1AO ATP synthases couple ion-transport of the AO sector and ATP synthesis/hydrolysis of the A3B3-headpiece via their stalk subunits D and F. Here, we produced and purified stable A3B3D- and A3B3DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Gö1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates (Vmax) of A3B3D and A3B3DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a Vmax of 7.9 s− 1 and 30.4 s− 1, respectively, while the KM is the same for both. Deletions of the N- or C-termini of subunit F abolished the effect of ATP hydrolysis activation. We generated subunit F mutant proteins with single amino acid substitutions and demonstrated that the subunit F residues S84 and R88 are important in stimulating ATP hydrolysis. Hybrid formation of the A3B3D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit ε of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis. NMRC (Natl Medical Research Council, S’pore) Accepted version

Specific ATP binding to the ε subunit of thermophilic F1-ATPase has been utilized for the biosensors of ATP in vivo. I report here that the ε subunit containing R103A/R115A mutations can bind ATP with a dissociation constant at 52 nM, which is two orders of magnitude higher affinity than the wild type. The mutant retained specificity for ATP; ADP and GTP bound to the mutant with dissociation constants 16 and 53 μM, respectively. Thus, the mutant would be a good platform for various types of nucleotide biosensor with appropriate modifications.

The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme's rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F1-catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ(0) cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.

F-ATP synthases are described to have mechanisms which regulate the unnecessary depletion of ATP pool during an energy limited state of the cell. Mg-ADP inhibition is one of the regulatory features where Mg-ADP gets entrapped in the catalytic site, preventing the binding of ATP and further inhibiting ATP hydrolysis. Knowledge about the existence and regulation of the related archaeal-type A1AO ATP synthases (A3B3CDE2FG2ac) is limited. We demonstrate MgADP inhibition of the enzymatically active A3B3D- and A3B3DF complexes of Methanosarcina mazei Gö1 A-ATP synthase and reveal the importance of the amino acids P235 and S238 inside the P-loop (GPFGSGKTV) of the catalytic A subunit. Substituting these two residues by the respective P-loop residues alanine and cysteine (GAFGCGKTV) of the related eukaryotic V-ATPase increases significantly the ATPase activity of the enzyme variant and abolishes MgADP inhibition. The atomic structure of the P235A, S238C double mutant of subunit A of the Pyrococcus horikoshii OT3 A-ATP synthase provides details of how these critical residues affect nucleotide-binding and ATP hydrolysis in this molecular engine. The qualitative data are confirmed by quantitative results derived from fluorescence correlation spectroscopy experiments. MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Accepted version

The F1FO-ATP synthase is the only enzyme in nature endowed with bi-functional catalytic mechanism of synthesis and hydrolysis of ATP. The enzyme functions, not only confined to energy transduction, are tied to three intrinsic features of the annular arrangement of c subunits which constitutes the so-called c-ring, the core of the membrane-embedded FO domain: (i) the c-ring constitution is linked to the number of ions (H(+) or Na(+)) channeled across the membrane during the dissipation of the transmembrane electrochemical gradient, which in turn determines the species-specific bioenergetic cost of ATP, the "molecular currency unit" of energy transfer in all living beings; (ii) the c-ring is increasingly involved in the mitochondrial permeability transition, an event linked to cell death and to most mitochondrial dysfunctions; (iii) the c subunit species-specific amino acid sequence and susceptibility to post-translational modifications can address antibacterial drug design according to the model of enzyme inhibitors which target the c subunits. Therefore, the simple c-ring structure not only allows the F1FO-ATP synthase to perform the two opposite tasks of molecular machine of cell life and death, but it also amplifies the enzyme's potential role as a drug target.

ATP synthases are molecular motors, which synthesize ATP, the ubiquitous energy source in all living cells. They use an electrochemical gradient to drive a rotation in the membrane embedded Fo domain, namely the c-ring, causing a conformational change in the soluble F1 domain which leads to the catalytic event. In the opposite fashion, they can also hydrolyse ATP to maintain the ion gradient across the membrane. To prevent wasteful ATP hydrolysis, bacteria and mammals have developed peculiar mechanistic features in addition to a common one, namely MgADP inhibition. Here I discuss the distinct ATPase inhibition mechanism in mitochondrial (IF1) and bacterial (subunits ε and ζ) F-type ATP synthases, based on available structural, biophysical and biochemical data.

Thecring of the Na+F1FoATP synthase from the anaerobic acetogenic bacteriumAcetobacterium woodiiis encoded by three different genes:atpE1,atpE2andatpE3. Subunitc1is similar to typical V-typecsubunits and has four transmembrane helices with one ion binding site. Subunitc2andc3are identical at the amino acid level and are typical F-typecsubunits with one ion binding site in two transmembrane helices. All three constitute a hybrid FoVocring, the first found in nature. To analyze whether other species may have similar hybrid rotors, we searched every genome sequence publicly available as of 23 February 2015 for F1FoATPase operons that have more than one gene encoding thecsubunit. This revealed no other species that has three differentcsubunit encoding genes but twelve species that encode one Fo- and one Vo-typecsubunit in one operon. Theircsubunits have the conserved binding motif for Na+. The organisms are all anaerobic. The advantage of hybridcrings for the organisms in their environments is discussed.

F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.

Like the evolutionary related F1FO ATP synthases and V1VO ATPases, the A1AO ATP synthases from archaea are multisubunit, membrane-bound transport machines that couple ion flow to the synthesis of ATP. Although the subunit composition is known for at least two species, nothing is known so far with respect to the function of individual subunits or amino acid residues. To pave the road for a functional analysis of A1AO ATP synthases, we have cloned the entire operon from Methanosarcina mazei into an expression vector and produced the enzyme in Escherichia coli. Inverted membrane vesicles of the recombinants catalyzed ATP synthesis driven by NADH oxidation as well as artificial driving forces. Δ μ ˜ H + as well as ΔpH were used as driving forces which is consistent with the inhibition of NADH-driven ATP synthesis by protonophores. Exchange of the conserved glutamate in subunit c led to a complete loss of ATP synthesis, proving that this residue is essential for H+ translocation. Exchange of two conserved arginine residues in subunit a has different effects on ATP synthesis. The role of these residues in ion translocation is discussed.

The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs) in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs.

Living organisms rely on the FoF1 ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the Fo motor uses a transmembrane electrochemical ion gradient to create clockwise torque that overcomes F1 ATPase-driven counterclockwise torque at high ATP is a major unresolved question. Using single FoF1 molecules embedded in lipid bilayer nanodiscs, we now report the observation of Fo-dependent rotation of the c10 ring in the ATP synthase (clockwise) direction against the counterclockwise force of ATPase-driven rotation that occurs upon formation of a leash with Fo stator subunit a. Mutational studies indicate that the leash is important for ATP synthase activity and support a mechanism in which residues aGlu-196 and cArg-50 participate in the cytoplasmic proton half-channel to promote leash formation.

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.

ATP synthases are remarkable proteins that regenerate the molecular fuel for cellular processes in all domains of life (1). Embedded into the inner membranes of mitochondria, chloroplasts, and bacteria, FoF1-ATP synthase produces most of the cellular ATP, using ADP and inorganic phosphate as inputs (Fig. 1 A ). This thermodynamically unfavorable reaction is powered by a proton electrochemical gradient across the membrane, which drives the rotation of the c-ring in the Fo part of ATP synthase (2). The asymmetrical central shaft connecting Fo and F1 then deforms the three active sites in the F1 part to catalyze ATP synthesis (1). The proper function of this machine requires efficient rotation of the c-ring in a highly viscous lipid membrane without any proton leaks. This problem is compounded by the unusual structure of the a-subunit facing the c-ring, with its long helices parallel to the membrane distorting the lipid packing around the Fo part (3, 4). Fig. 1. Schematic of FoF1-ATP synthase function. ( A , Top ) Proton translocation mediated by the c-ring of the membrane-embedded Fo part drives rotation. ( A , Bottom ) The asymmetrically shaped rotor axis attached to the c-ring distorts the active sites in the F1 part, where ATP is synthesized from ADP and inorganic phosphate Pi. Simulations by Duncan et al. (5) show that CL, an abundant lipid in the inner mitochondrial membrane, accumulates near the c-ring. The structure is based on Protein Data Bank ID code 5FIK (4). ( B ) CL is an unusual lipid with four acyl chains. In PNAS, Duncan et al. (5) report that an unusual lipid–protein interaction in the Fo part of ATP synthase is central to efficient … [↵][1]1To whom correspondence should be addressed. Email: gerhard.hummer{at}biophys.mpg.de. [1]: #xref-corresp-1-1

Mitochondrial FoF1ATP synthase is a membrane bound molecular machine central to cellular energy conversion and cristae architecture. Recently, a novel domain has been visualized in the intermembrane space region of mammalian ATP synthase. The complete three-dimensional (3D) structure, composition and function of this domain - which we term intermembrane space domain (IMD) - are unknown. Here, we present two distinct 3D structures of monomeric bovine FoF1ATP synthase by single particle cryo-electron microscopy (cryo-EM) that differ by the presence and absence of the IMD. Comparison of both structures reveals the IMD to be a bipartite and weakly associated domain of FoF1ATP synthase. The tubular sub-domain of the IMD appears to contact the rotor-ring region, its globular sub-domain is anchored in the membrane-bending kink of the ATP synthase. However, absence of the IMD does not impact the kink in the transmembrane region ruling out a functional role in membrane bending. By combining our structural analysis with chemical cross-linking and reported biochemical, genetic and structural data we identify 6.8PL and DAPIT as the subunits forming the intermembrane space domain. We compare the present structure of the mammalian IMD in the bovine FoF1ATP synthase monomer with structures of dimeric FoF1ATP synthase from yeast and ciliate showing that the IMD is a common, but structurally divergent feature of several mitochondrial ATP synthases. On the basis of our analysis we discuss potential functions of the novel domain in rotary catalysis, oligomerization and mitochondrial permeability transition.

The opening of a non-specific channel, known as the permeability transition pore (PTP), in the inner membranes of mitochondria, can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane and ATP synthesis, and cell death. Pore opening can be inhibited by cyclosporin A mediated via cyclophilin D. It has been proposed that the pore is associated with the dimeric ATP synthase, and that the OSCP (oligomycin sensitivity conferral protein), a component of the enzyme’s peripheral stalk, provides the site where cyclophilin D interacts. Subunit b contributes a central α-helical structure to the peripheral stalk, extending from near the top of the enzyme’s catalytic domain and crossing the membrane domain of the enzyme via two α-helices. We investigated the possible involvement of the subunit b and the OSCP in the PTP by generating clonal cells, HAP1-Δb and HAP1-ΔOSCP, lacking the membrane domain of subunit b or the OSCP, respectively, in which the correponding genes ATP5F1 and ATP5O had been disrupted. Both cell lines preserve the characteristic properties of the PTP. Therefore, the membrane domain of subunit b does not contribute to the PTP, and the OSCP does not provide the site of interaction with cyclophilin D. The membrane subunits ATP6, ATP8 and subunit c have been eliminated previously from possible participation in the PTP. Therefore, the only subunits of ATP synthase that could participate in pore formation are e, f, g, DAPIT (diabetes associated protein in insulin sensitive tissues) and the 6.8 kDa proteolipid.

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases. DOI: http://dx.doi.org/10.7554/eLife.10180.001, eLife digest A molecule called adenosine triphosphate (ATP) is the energy currency in cells. Most of the ATP used by cells is made by the membrane-embedded enzyme ATP synthase. This enzyme is found in membranes inside specialized compartments known as mitochondria. ATP synthase is made up of many protein subunits that work together as a molecular machine. Hydrogen ions flow across the membrane through the ATP synthase, turning a rotor structure within the enzyme, which leads to the production of ATP. It is not known how the transport of hydrogen ions causes rotation of the rotor. Some researchers have proposed that the enzyme works as a ratchet that is driven by the random Brownian motion of the rotor. That is, the rotational position of the rotor fluctuates randomly, but a ratchet mechanism ensures that there is a net rotation in one direction. However, there is currently little experimental evidence to back up this theory, which is known as the Brownian ratchet model. Zhou, Rohou et al. used a technique called electron cryomicroscopy (or cryo-EM) to study ATP synthase from cows. The cryo-EM data made it possible to use computer software to construct a three-dimensional model of the enzyme that is more detailed than previous attempts. Zhou, Rohou et al. show that the structure of ATP synthase is flexible, with the different protein subunits bending, flexing, and rotating relative to each other. This variability in the position of the rotor is consistent with the Brownian ratchet model. Together, these findings reveal important new details about the structure of ATP synthase and provide some of the first experimental evidence for the Brownian ratchet model. The new three-dimensional structure of ATP synthase will open the door to testing hypotheses of how the ATP synthase works. DOI: http://dx.doi.org/10.7554/eLife.10180.002

We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis.

F‐ATP synthases convert the electrochemical energy of the H+ gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F‐ATP synthases can also undergo a Ca2+‐dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca2+ binding site and the mechanism(s) through which Ca2+ can transform the energy‐conserving enzyme into a dissipative structure promoting cell death remain unknown. Through in vitro, in vivo and in silico studies we (i) pinpoint the “Ca2+‐trigger site” of the PTP to the catalytic site of the F‐ATP synthase β subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the β subunit, which show a selective decrease in Ca2+‐ATP hydrolysis, confer resistance to Ca2+‐induced, PTP‐dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F‐ATP synthase to a channel and of its role in cell death.

The epsilon subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the e subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the e subunit from bacterial ATP synthases.

This paper describes the role of α-subunit VISIT-DG sequence residue αThr-349 in the catalytic sites of Escherichia coli F1Fo ATP synthase. X-ray structures show the highly conserved αThr-349 in the proximity (2.68 Å) of the conserved phosphate binding residue βR182 in the phosphate binding subdomain. αT349A, -D, -Q, and -R mutations caused 90-100-fold losses of oxidative phosphorylation and reduced ATPase activity of F1Fo in membranes. Double mutation αT349R/βR182A was able to partially compensate for the absence of known phosphate binding residue βR182. Azide, fluoroaluminate, and fluoroscandium caused insignificant inhibition of αT349A, -D, and -Q mutants, slight inhibition of the αT349R mutant, partial inhibition of the αT349R/βR182A double mutant, and complete inhibition of the wild type. Whereas NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) inhibited wild-type ATPase and its αT349A, -D, -R, and -Q mutants essentially completely, βR182A ATPase and double mutant αT349A/βR182A were inhibited partially. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in the wild type, αT349R, and double mutant αT349R/βR182A but not in αT349A, αT349D, or αT349Q. The results demonstrate that αThr-349 is a supplementary residue involved in phosphate binding and transition state stabilization in ATP synthase catalytic sites through its interaction with βR182.

Vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump. The proton pump is essential for the regulation of pH in various eukaryotic cellular processes. Among the 14 subunits that constitute V-ATPase, d subunit mediates coupling between cytosolic and membrane domains. Whereas d1 is expressed ubiquitously in various types of cells, its isoform d2 is only expressed in specific cells or tissues. To characterize these isoforms, we expressed and purified the isoforms of human V-ATPase d subunits using Escherichia coli over-expression system. Subunit d1 and d2 were purified as homogeneous monomers as demonstrated by dynamic light scattering (DLS) analysis. Secondary structures of d subunits were estimated to be composed of 73% α-helix and 2% β-sheet, as analyzed using circular dichroism (CD) analysis. Although sequence identity and secondary structures of d subunits were highly similar, the relative stability against thermal stress was higher for d1 than d2. Efficient expression and purification of d subunits, together with biophysical and biochemical characterization, presented in this study is expected to facilitate further structural analysis to clarify specific inter-molecular interactions involved in multi-subunit assembly and regulation of H+ transporters.

F-1-ATPase (F-1) is the catalytic portion of ATP synthase, a rotary motor protein that couples proton gradients to ATP synthesis. Driven by a proton flux, the F, asymmetric gamma subunit undergoes a stepwise rotation inside the alpha(3)beta(3) headpiece and causes the beta subunits' binding sites to cycle between states of different affinity for nucleotides. These concerted transitions drive the synthesis of ATP from ADP and phosphate. Here, we study the coupling between the mechanical progression of gamma and the conformations of alpha(3)beta(3). Using molecular dynamics simulations, we show that the nucleotide-free beta subunit, initially in the open, low-affinity state, undergoes a spontaneous closing transition to the half-open state in response to the gamma rotation in the synthesis direction. We estimate the kinetics of this spontaneous conformational change and analyze its mechanism and driving forces. By computing free energy profiles, we find that the isolated empty beta subunit preferentially adopts the half-open conformation and that the transition to this conformation from the fully open state is accompanied by well-defined changes in the structure and interactions of the active site region. These results suggest that ADP binding to F, occurs via conformational selection and is preceded by the transition of the active site to the half-open conformation, driven by the intrinsic elasticity of beta. Our results also indicate that opening of the nucleotide-free beta during hydrolysis is not spontaneous, as previously assumed. Rather, the fully open conformation observed in the F-1 X-ray structure is enforced sterically by the gamma subunit whose orientation is stabilized by interactions with the two other beta subunits in the completely closed state. This finding supports the notion that gamma acts by coupling the extreme conformational states of beta subunits within the alpha(3)beta(3) hexamer and therefore is responsible for high efficiency of the coordinated catalysis.

The central stalk of the ATP synthase is an elongated hetero-oligomeric structure providing a physical connection between the catalytic sites in F1and the proton translocation channel in F0for energy transduction between the two subdomains. The shape of the central stalk and relevance to energy coupling are essentially the same in ATP synthases from all forms of life, yet the protein composition of this domain changed during evolution of the mitochondrial enzyme from a two- to a three-subunit structure (γ, δ, ε). Whereas the mitochondrial γ- and δ-subunits are homologues of the bacterial central stalk proteins, the deliberate addition of subunit ε is poorly understood. Here we report that down-regulation of the gene (ATP15) encoding the ε-subunit rapidly leads to lethal F0-mediated proton leaks through the membrane because of the loss of stability of the ATP synthase. The ε-subunit is thus essential for oxidative phosphorylation. Moreover, mutations in F0subunits a and c, which slow the proton translocation rate, are identified that prevent ε-deficient ATP synthases from dissipating the electrochemical potential. Cumulatively our data lead us to propose that the ε-subunit evolved to permit operation of the central stalk under the torque imposed at the normal speed of proton movement through mitochondrial F0.

Dysfunction of TMEM70 disrupts the biogenesis of ATP synthase and represents the frequent cause of autosomal recessive encephalocardiomyopathy. We used tagged forms of TMEM70 and demonstrated that it has a hairpin structure with the N- and C-termini oriented towards the mitochondrial matrix. On BN-PAGE TMEM70 was detected in multiple forms including dimers and displayed partial overlap with assembled ATP synthase. Immunoprecipitation studies confirmed mutual interactions between TMEM70 molecules but, together with immunogold electron microscopy, not direct interaction with ATP synthase subunits. This indicates that the biological function of TMEM70 in the ATP synthase biogenesis may be mediated through interaction with other protein(s).

ATP synthases (FoF1-ATPases) of chloroplasts, mitochondria, and bacteria catalyze ATP synthesis or hydrolysis coupled with the transmembrane transfer of protons or sodium ions. Their activity is regulated through their reversible inactivation resulting from a decreased transmembrane potential difference. The inactivation is believed to conserve ATP previously synthesized under conditions of sufficient energy supply against unproductive hydrolysis. This review is focused on the mechanism of nucleotide-dependent regulation of the ATP synthase activity where the so-called noncatalytic nucleotide binding sites are involved. Properties of these sites varying upon free enzyme transition to its membrane-bound form, their dependence on membrane energization, and putative mechanisms of noncatalytic site-mediated regulation of the ATP synthase activity are discussed.

Significance: Regulation of the activity of the chloroplast ATP synthase is largely accomplished by the chloroplast thioredoxin system, the main redox regulation system in chloroplasts, which is directly coupled to the photosynthetic reaction. We review the current understanding of the redox regulation system of the chloroplast ATP synthase. Recent Advances: The thioredoxin-targeted portion of the ATP synthase consists of two cysteines located on the central axis subunit γ. The redox state of these two cysteines is under the influence of chloroplast thioredoxin, which directly controls rotation during catalysis by inducing a conformational change in this subunit. The molecular mechanism of redox regulation of the chloroplast ATP synthase has recently been determined. Critical Issues: Regulation of the activity of the chloroplast ATP synthase is critical in driving efficiency into the ATP synthesis reaction in chloroplasts. Future Directions: The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system. Antioxid. Redox Signal. 19, 1846–1854.

The ATP synthase (F o F 1 ) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane by F o to ATP synthesis or hydrolysis in F 1 . Whereas good knowledge of the nanostructure and the rotary mechanism of the ATP synthase is at hand, the assembly pathway of the 22 polypeptide chains present in a stoichiometry of ab 2 c 10 α 3 β 3 γδϵ has so far not received sufficient attention. In our studies, mutants that synthesize different sets of F o F 1 subunits allowed the characterization of individually formed stable subcomplexes. Furthermore, the development of a time-delayed in vivo assembly system enabled the subsequent synthesis of particular missing subunits to allow the formation of functional ATP synthase complexes. These observations form the basis for a model that describes the assembly pathway of the E. coli ATP synthase from pre-formed subcomplexes, thereby avoiding membrane proton permeability by a concomitant assembly of the open H + -translocating unit within a coupled F o F 1 complex.

The ATP synthase (FOF1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of ATP. This nanomotor is composed of the rotor c10γϵ and the stator ab2α3β3δ. To study the assembly of this multimeric enzyme complex consisting of membrane-integral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled FOF1 complexes by affinity chromatography with the use of mutants synthesizing different sets of FOF1 subunits. Together with a time-delayed in vivo assembly system, the results demonstrate that FOF1 is assembled in a modular way via subcomplexes, thereby preventing the formation of a functional H+-translocating unit as intermediate product. Surprisingly, during the biogenesis of FOF1, F1 subunit δ is the key player in generating stable FO. Subunit δ serves as clamp between ab2 and c10α3β3γϵ and guarantees that the open H+ channel is concomitantly assembled within coupled FOF1 to maintain the low membrane proton permeability essential for viability, a general prerequisite for the assembly of multimeric H+-translocating enzymes. Background: FOF1 ATP synthases are rotary nanomachines transporting protons across the membrane during catalysis. Results: Escherichia coli FOF1 is assembled from subcomplexes with subunit δ functioning as clamp to generate stable FO. Conclusion: δ guarantees that the open proton channel is concomitantly assembled within coupled FOF1. Significance: We investigate how a proton-translocating unit is assembled while simultaneously maintaining the low membrane proton permeability essential for viability.

Escherichia coli F O F 1 ATP synthase, a rotary nanomachine, is composed of eight different subunits in a α 3 β 3 γδe ab 2 c 10 stoichiometry. Whereas F O F 1 has been studied in detail with regard to its structure and function, much less is known about how this multisubunit enzyme complex is assembled. Single-subunit atp deletion mutants are known to be arrested in assembly, thus leading to formation of partially assembled subcomplexes. To determine whether those subcomplexes are preserved in a stable standby mode, a time-delayed in vivo assembly system was developed. To establish this approach, we targeted the time-delayed assembly of membrane-integrated subunit a into preformed F O F 1 lacking subunit a (F O F 1 - a ) which is known to form stable subcomplexes in vitro . Two expression systems ( araBADp and T7 p-laco ) were adjusted to provide compatible, mutually independent, and sufficiently stringent induction and repression regimens. In detail, all structural atp genes except atpB (encoding subunit a ) were expressed under the control of araBADp and induced by arabinose. Following synthesis of F O F 1 - a during growth, expression was repressed by glucose/d-fucose, and degradation of atp mRNA controlled by real-time reverse transcription-PCR. A time-delayed expression of atpB under T7 p-laco control was subsequently induced in trans by addition of isopropyl-β-d-thiogalactopyranoside. Formation of fully assembled, and functional, F O F 1 complexes was verified. This demonstrates that all subunits of F O F 1 - a remain in a stable preformed state capable to integrate subunit a as the last subunit. The results reveal that the approach presented here can be applied as a general method to study the assembly of heteromultimeric protein complexes in vivo .

The chloroplast CF0-CF1-ATP synthase (ATP synthase) is activated in the light and inactivated in the dark by thioredoxin-mediated redox modulation of a disulfide bridge on its γ subunit. The activity of the ATP synthase is also fine-tuned during steady-state photosynthesis in response to metabolic changes, e.g. altering CO2 levels to adjust the thylakoid proton gradient and thus the regulation of light harvesting and electron transfer. The mechanism of this fine-tuning is unknown. We test here the possibility that it also involves redox modulation. We found that modifying the Arabidopsis thaliana γ subunit by mutating three highly conserved acidic amino acids, D211V, E212L, and E226L, resulted in a mutant, termed mothra, in which ATP synthase which lacked light-dark regulation had relatively small effects on maximal activity in vivo. In situ equilibrium redox titrations and thiol redox-sensitive labeling studies showed that the γ subunit disulfide/sulfhydryl couple in the modified ATP synthase has a more reducing redox potential and thus remains predominantly oxidized under physiological conditions, implying that the highly conserved acidic residues in the γ subunit influence thiol redox potential. In contrast to its altered light-dark regulation, mothra retained wild-type fine-tuning of ATP synthase activity in response to changes in ambient CO2 concentrations, indicating that the light-dark- and metabolic-related regulation occur through different mechanisms, possibly via small molecule allosteric effectors or covalent modification.

The molecular mechanism by which the membrane-embedded FO sector of the mitochondrial ATP synthase translocates protons, thus dissipating the transmembrane protonmotive force and leading to ATP synthesis, involves the neutralization of the carboxylate residues of the c-ring. Carboxylates are thought to constitute the binding sites for ion translocation. In order to cast light on this mechanism, we exploited N,N’-dicyclohexylcarbodiimide, which covalently binds to FO c-ring carboxylates, and ionophores which selectively modulate the transmembrane electric (Δφ) and chemical (ΔpH) gradients such as valinomycin, nigericin and dinitrophenol. ATP hydrolysis was evaluated in mitochondrial preparations and/or inside-out submitochondrial particles from mussel and mammalian tissues under different experimental conditions. The experiments pointed out striking similarities between mussel and mammalian mitochondrial ATP synthase. Our results support the hypothesis that the ATP synthase of Mytilus galloprovincialis induces intersubunit torque generation and translocates H+ by coordinating the hydronium ion (H3O+) in the ion binding site of FO. Our results are consistent with the hypothesis that in mussel mitochondria the main component of the electrochemical gradient driving proton flux and ATP synthesis is Δφ. Therefore, mussel FO probably contains a small c-ring, which implies a low bioenergetic cost of making ATP as in mammals. These features which make mussel mitochondria as efficient in ATP production as mammalian ones may be especially advantageous in facultative aerobic species which intermittently exploit mitochondrial respiration to generate ATP.

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1.

The subunit ε of bacterial F 1 F O ATP synthases plays an important regulatory role in coupling and catalysis via conformational transitions of its C-terminal domain. Here we present the first low-resolution solution structure of ε of Mycobacterium tuberculosis ( Mt ε) F 1 F O ATP synthase and the nuclear magnetic resonance (NMR) structure of its C-terminal segment ( Mt ε 103–120 ). Mt ε is significantly shorter (61.6 Å) than forms of the subunit in other bacteria, reflecting a shorter C-terminal sequence, proposed to be important in coupling processes via the catalytic β subunit. The C-terminal segment displays an α-helical structure and a highly positive surface charge due to the presence of arginine residues. Using NMR spectroscopy, fluorescence spectroscopy, and mutagenesis, we demonstrate that the new tuberculosis (TB) drug candidate TMC207, proposed to bind to the proton translocating c -ring, also binds to Mt ε. A model for the interaction of TMC207 with both ε and the c -ring is presented, suggesting that TMC207 forms a wedge between the two rotating subunits by interacting with the residues W15 and F50 of ε and the c -ring, respectively. T19 and R37 of ε provide the necessary polar interactions with the drug molecule. This new model of the mechanism of TMC207 provides the basis for the design of new drugs targeting the F 1 F O ATP synthase in M. tuberculosis .

Mitochondrial F(1)F(o) ATP synthase is an enzymatic complex involved in the aerobic synthesis of ATP. It is well known that several enzymes are organized in supramolecular complexes in the inner mitochondrial membrane. The ATP synthase supramolecular assembly is mediated through two interfaces. One leads to dimer formation and the other to oligomer formation. In yeast, the presence of ATP synthase oligomers has been described as essential to the maintenance of the mitochondrial cristae ultrastructure. Indeed, the destabilization of the interactions between monomers was shown to alter the organization of the inner mitochondrial membrane, leading to the formation of onion-like structures similar to those observed in some mitochondrial pathologies. By using information obtained this decade (structure modeling, electron microscopy and cross-linking), this paper (i) reviews the actual state of the art and (ii) proposes a topological model of the transmembrane domains and interfaces of the ATP synthase's tetramer. This review also discusses the physiological role of this oligomerization process and its potential implications in mammal pathology. This article is part of a Directed Issue entitled: Bioenergetic Dysfunction, adaptation and therapy.

ATP synthase is a biological motor that produces a molecule called adenosine tri-phosphate (ATP for short), which acts as the major store of chemical energy in cells. A single molecule of ATP contains three phosphate groups: the cell can remove one of these phosphates to make a molecule called adenosine di-phosphate (ADP) and release energy to drive a variety of biological processes. ATP synthase sits in the membranes that separate cell compartments or form barriers around cells. When cells break down food they transport hydrogen ions across these membranes so that each side of the membrane has a different level (or “concentration”) of hydrogen ions. Movement of hydrogen ions from an area with a high concentration to a low concentration causes ATP synthase to rotate like a turbine. This rotation of the enzyme results in ATP synthase adding a phosphate group to ADP to make a new molecule of ATP. In certain conditions cells need to switch off the ATP synthase and this is done by changing the shape of the central shaft in a process called autoinhibition, which blocks the rotation. The ATP synthase from a bacterium known as E. coli – which is commonly found in the human gut –has been used as a model to study how this biological motor works. However, since the precise details of the three-dimensional structure of ATP synthase have remained unclear it has been difficult to interpret the results of these studies. Sobti et al. used a technique called Cryo-electron microscopy to investigate the structure of ATP synthase from E. coli. This made it possible to develop a three-dimensional model of the ATP synthase in its autoinhibited form. The structural data could also be split into three distinct shapes that relate to dwell points in the rotation of the motor where the rotation has been inhibited. These models further our understanding of ATP synthases and provide a template to understand the findings of previous studies. Further work will be needed to understand this essential biological process at the atomic level in both its inhibited and uninhibited form. This will reveal the inner workings of a marvel of the natural world and may also lead to the discovery of new antibiotics against related bacteria that cause diseases in humans.

F1Fo-ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H+ flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H+ within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F1 and FO domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo.

The ATP synthase is a molecular rotor that recycles ADP into ATP. Leone and Faraldo-Gómez use structural modeling to reinterpret and reconcile recent cryo-EM data for its membrane domain with other experimental evidence, gaining insights into its mechanism and the mode of inhibition by oligomycin., Two subunits within the transmembrane domain of the ATP synthase—the c-ring and subunit a—energize the production of 90% of cellular ATP by transducing an electrochemical gradient of H+ or Na+ into rotational motion. The nature of this turbine-like energy conversion mechanism has been elusive for decades, owing to the lack of definitive structural information on subunit a or its c-ring interface. In a recent breakthrough, several structures of this complex were resolved by cryo–electron microscopy (cryo-EM), but the modest resolution of the data has led to divergent interpretations. Moreover, the unexpected architecture of the complex has cast doubts on a wealth of earlier biochemical analyses conducted to probe this structure. Here, we use quantitative molecular-modeling methods to derive a structure of the a–c complex that is not only objectively consistent with the cryo-EM data, but also with correlated mutation analyses of both subunits and with prior cross-linking and cysteine accessibility measurements. This systematic, integrative approach reveals unambiguously the topology of subunit a and its relationship with the c-ring. Mapping of known Cd2+ block sites and conserved protonatable residues onto the structure delineates two noncontiguous pathways across the complex, connecting two adjacent proton-binding sites in the c-ring to the space on either side of the membrane. The location of these binding sites and of a strictly conserved arginine on subunit a, which serves to prevent protons from hopping between them, explains the directionality of the rotary mechanism and its strict coupling to the proton-motive force. Additionally, mapping of mutations conferring resistance to oligomycin unexpectedly reveals that this prototypical inhibitor may bind to two distinct sites at the a–c interface, explaining its ability to block the mechanism of the enzyme irrespective of the direction of rotation of the c-ring. In summary, this study is a stepping stone toward establishing the mechanism of the ATP synthase at the atomic level.