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.

Mitochondrial Sirtuin Network Reveals Dynamic SIRT3-Dependent Deacetylation in Response to Membrane Depolarization.

Mitochondrial sirtuins, SIRT3-5, are NAD+-dependent deacylases and ADP-ribosyltransferases that are critical for stress responses. However, a comprehensive understanding of sirtuin targets, regulation of sirtuin activity, and the relationships between sirtuins remains a key challenge in mitochondrial physiology. Here, we employ systematic interaction proteomics to elucidate the mitochondrial sirtuin protein interaction landscape. This work reveals sirtuin interactions with numerous functional modules within mitochondria, identifies candidate sirtuin substrates, and uncovers a fundamental role for sequestration of SIRT3 by ATP synthase in mitochondrial homeostasis. In healthy mitochondria, a pool of SIRT3 binds ATP synthase, but upon matrix pH reduction with concomitant loss of mitochondrial membrane potential, SIRT3 dissociates. This release correlates with rapid deacetylation of matrix proteins, and SIRT3 is required for recovery of membrane potential. In vitro reconstitution experiments, as well as analysis of CRISPR/Cas9-engineered cells, indicate that pH-dependent SIRT3 release requires H135 in the ATP5O subunit of ATP synthase. Our SIRT3-5 interaction network provides a framework for discovering novel biological functions regulated by mitochondrial sirtuins.

Structure of the human ATP synthase.

Biological energy currency ATP is produced by F1Fo-ATP synthase. However, the molecular mechanism for human ATP synthase action remains unknown. Here, we present snapshot images for three main rotational states and one substate of human ATP synthase using cryoelectron microscopy. These structures reveal that the release of ADP occurs when the ß subunit of F1Fo-ATP synthase is in the open conformation, showing how ADP binding is coordinated during synthesis. The accommodation of the symmetry mismatch between F1 and Fo motors is resolved by the torsional flexing of the entire complex, especially the γ subunit, and the rotational substep of the c subunit. Water molecules are identified in the inlet and outlet half-channels, suggesting that the proton transfer in these two half-channels proceed via a Grotthus mechanism. Clinically relevant mutations are mapped to the structure, showing that they are mainly located at the subunit-subunit interfaces, thus causing instability of the complex.

Unique Properties of Apicomplexan Mitochondria.

Apicomplexan parasites constitute more than 6,000 species infecting a wide range of hosts. These include important pathogens such as those causing malaria and toxoplasmosis. Their evolutionary emergence coincided with the dawn of animals. Mitochondrial genomes of apicomplexan parasites have undergone dramatic reduction in their coding capacity, with genes for only three proteins and ribosomal RNA genes present in scrambled fragments originating from both strands. Different branches of the apicomplexans have undergone rearrangements of these genes, with Toxoplasma having massive variations in gene arrangements spread over multiple copies. The vast evolutionary distance between the parasite and the host mitochondria has been exploited for the development of antiparasitic drugs, especially those used to treat malaria, wherein inhibition of the parasite mitochondrial respiratory chain is selectively targeted with little toxicity to the host mitochondria. We describe additional unique characteristics of the parasite mitochondria that are being investigated and provide greater insights into these deep-branching eukaryotic pathogens.

Mechanism of mycobacterial ATP synthase inhibition by squaramides and second generation diarylquinolines.

Mycobacteria, such as Mycobacterium tuberculosis, depend on the activity of adenosine triphosphate (ATP) synthase for growth. The diarylquinoline bedaquiline (BDQ), a mycobacterial ATP synthase inhibitor, is an important medication for treatment of drug-resistant tuberculosis but suffers from off-target effects and is susceptible to resistance mutations. Consequently, both new and improved mycobacterial ATP synthase inhibitors are needed. We used electron cryomicroscopy and biochemical assays to study the interaction of Mycobacterium smegmatis ATP synthase with the second generation diarylquinoline TBAJ-876 and the squaramide inhibitor SQ31f. The aryl groups of TBAJ-876 improve binding compared with BDQ, while SQ31f, which blocks ATP synthesis ~10 times more potently than ATP hydrolysis, binds a previously unknown site in the enzyme's proton-conducting channel. Remarkably, BDQ, TBAJ-876, and SQ31f all induce similar conformational changes in ATP synthase, suggesting that the resulting conformation is particularly suited for drug binding. Further, high concentrations of the diarylquinolines uncouple the transmembrane proton motive force while for SQ31f they do not, which may explain why high concentrations of diarylquinolines, but not SQ31f, have been reported to kill mycobacteria.

Mechanism of proton-powered c-ring rotation in a mitochondrial ATP synthase.

Proton-powered c-ring rotation in mitochondrial ATP synthase is crucial to convert the transmembrane protonmotive force into torque to drive the synthesis of adenosine triphosphate (ATP). Capitalizing on recent cryo-EM structures, we aim at a structural and energetic understanding of how functional directional rotation is achieved. We performed multi-microsecond atomistic simulations to determine the free energy profiles along the c-ring rotation angle before and after the arrival of a new proton. Our results reveal that rotation proceeds by dynamic sliding of the ring over the a-subunit surface, during which interactions with conserved polar residues stabilize distinct intermediates. Ordered water chains line up for a Grotthuss-type proton transfer in one of these intermediates. After proton transfer, a high barrier prevents backward rotation and an overall drop in free energy favors forward rotation, ensuring the directionality of c-ring rotation required for the thermodynamically disfavored ATP synthesis. The essential arginine of the a-subunit stabilizes the rotated configuration through a salt bridge with the c-ring. Overall, we describe a complete mechanism for the rotation step of the ATP synthase rotor, thereby illuminating a process critical to all life at atomic resolution.

The mitochondrial ATP synthase is a negative regulator of the mitochondrial permeability transition pore.

The mitochondrial permeability transition pore (mPTP) is a channel in the inner mitochondrial membrane whose sustained opening in response to elevated mitochondrial matrix Ca2+ concentrations triggers necrotic cell death. The molecular identity of mPTP is unknown. One proposed candidate is the mitochondrial ATP synthase, whose canonical function is to generate most ATP in multicellular organisms. Here, we present mitochondrial, cellular, and in vivo evidence that, rather than serving as mPTP, the mitochondrial ATP synthase inhibits this pore. Our studies confirm previous work showing persistence of mPTP in HAP1 cell lines lacking an assembled mitochondrial ATP synthase. Unexpectedly, however, we observe that Ca2+-induced pore opening is markedly sensitized by loss of the mitochondrial ATP synthase. Further, mPTP opening in cells lacking the mitochondrial ATP synthase is desensitized by pharmacological inhibition and genetic depletion of the mitochondrial cis-trans prolyl isomerase cyclophilin D as in wild-type cells, indicating that cyclophilin D can modulate mPTP through substrates other than subunits in the assembled mitochondrial ATP synthase. Mitoplast patch clamping studies showed that mPTP channel conductance was unaffected by loss of the mitochondrial ATP synthase but still blocked by cyclophilin D inhibition. Cardiac mitochondria from mice whose heart muscle cells we engineered deficient in the mitochondrial ATP synthase also demonstrate sensitization of Ca2+-induced mPTP opening and desensitization by cyclophilin D inhibition. Further, these mice exhibit strikingly larger myocardial infarctions when challenged with ischemia/reperfusion in vivo. We conclude that the mitochondrial ATP synthase does not function as mPTP and instead negatively regulates this pore.

Molecular mechanism and energetics of coupling between substrate binding and product release in the F1-ATPase catalytic cycle.

F1-ATPase is a motor protein that couples the rotation of its rotary [Formula: see text] subunit with ATP synthesis or hydrolysis. Single-molecule experiments indicate that nucleotide binding and release events occur almost simultaneously during the synthesis cycle, allowing the energy gain due to spontaneous binding of ADP to one catalytic [Formula: see text] subunit to be directly harnessed for driving the release of ATP from another rather than being dissipated as heat. Here, we examine the unknown mechanism of this coupling that is critical for an exceptionally high mechanochemical efficiency of F1-ATPase by means of all-atom free-energy simulations. We find that nondissipative and kinetically fast progression of the motor in the synthesis direction requires a concerted conformational change involving the closure of the ADP-binding [Formula: see text] subunit followed by the gradual opening of the ATP-releasing [Formula: see text] subunit over the course of the 30 to 40° rotary substep of the [Formula: see text] subunit. This rotary substep, preceding the ATP-dependent metastable state, allows for the recovery of a large portion of the ADP binding energy in the conformation of ATP-bound [Formula: see text] that gradually adopts the low-affinity conformation, captured also by the recent cryo-EM structure of this elusive state. The release of ATP from this nearly open conformation leads to its further opening, which enables the progression of the motor to the next catalytic metastable state. Our simulations explain this energy conversion mechanism in terms of intersubunit and ligand-protein interactions.

Electron transport chains as a window into the earliest stages of evolution.

The origin and early evolution of life is generally studied under two different paradigms: bottom up and top down. Prebiotic chemistry and early Earth geochemistry allow researchers to explore possible origin of life scenarios. But for these "bottom-up" approaches, even successful experiments only amount to a proof of principle. On the other hand, "top-down" research on early evolutionary history is able to provide a historical account about ancient organisms, but is unable to investigate stages that occurred during and just after the origin of life. Here, we consider ancient electron transport chains (ETCs) as a potential bridge between early evolutionary history and a protocellular stage that preceded it. Current phylogenetic evidence suggests that ancestors of several extant ETC components were present at least as late as the last universal common ancestor of life. In addition, recent experiments have shown that some aspects of modern ETCs can be replicated by minerals, protocells, or organic cofactors in the absence of biological proteins. Here, we discuss the diversity of ETCs and other forms of chemiosmotic energy conservation, describe current work on the early evolution of membrane bioenergetics, and advocate for several lines of research to enhance this understanding by pairing top-down and bottom-up approaches.

Two specific domains of the γ subunit of chloroplast FoF1 provide redox regulation of the ATP synthesis through conformational changes.

Chloroplast FoF1-ATP synthase (CFoCF1) converts proton motive force into chemical energy during photosynthesis. Although many studies have been done to elucidate the catalytic reaction and its regulatory mechanisms, biochemical analyses using the CFoCF1 complex have been limited because of various technical barriers, such as the difficulty in generating mutants and a low purification efficiency from spinach chloroplasts. By taking advantage of the powerful genetics available in the unicellular green alga Chlamydomonas reinhardtii, we analyzed the ATP synthesis reaction and its regulation in CFoCF1. The domains in the γ subunit involved in the redox regulation of CFoCF1 were mutated based on the reported structure. An in vivo analysis of strains harboring these mutations revealed the structural determinants of the redox response during the light/dark transitions. In addition, we established a half day purification method for the entire CFoCF1 complex from C. reinhardtii and subsequently examined ATP synthesis activity by the acid-base transition method. We found that truncation of the ß-hairpin domain resulted in a loss of redox regulation of ATP synthesis (i.e., constitutively active state) despite retaining redox-sensitive Cys residues. In contrast, truncation of the redox loop domain containing the Cys residues resulted in a marked decrease in the activity. Based on this mutation analysis, we propose a model of redox regulation of the ATP synthesis reaction by the cooperative function of the ß-hairpin and the redox loop domains specific to CFoCF1.

ROS signaling-induced mitochondrial Sgk1 expression regulates epithelial cell renewal.

Many types of differentiated cells can reenter the cell cycle upon injury or stress. The underlying mechanisms are still poorly understood. Here, we investigated how quiescent cells are reactivated using a zebrafish model, in which a population of differentiated epithelial cells are reactivated under a physiological context. A robust and sustained increase in mitochondrial membrane potential was observed in the reactivated cells. Genetic and pharmacological perturbations show that elevated mitochondrial metabolism and ATP synthesis are critical for cell reactivation. Further analyses showed that elevated mitochondrial metabolism increases mitochondrial ROS levels, which induces Sgk1 expression in the mitochondria. Genetic deletion and inhibition of Sgk1 in zebrafish abolished epithelial cell reactivation. Similarly, ROS-dependent mitochondrial expression of SGK1 promotes S phase entry in human breast cancer cells. Mechanistically, SGK1 coordinates mitochondrial activity with ATP synthesis by phosphorylating F1Fo-ATP synthase. These findings suggest a conserved intramitochondrial signaling loop regulating epithelial cell renewal.

Proton gradient across the chloroplast thylakoid membrane governs the redox regulatory function of ATP synthase.

Chloroplast ATP synthase (CFoCF1) synthesizes ATP by using a proton electrochemical gradient across the thylakoid membrane, termed ΔµH+, as an energy source. This gradient is necessary not only for ATP synthesis but also for reductive activation of CFoCF1 by thioredoxin, using reducing equivalents produced by the photosynthetic electron transport chain. ΔµH+ comprises two thermodynamic components: pH differences across the membrane (ΔpH) and the transmembrane electrical potential (ΔΨ). In chloroplasts, the ratio of these two components in ΔµH+ is crucial for efficient solar energy utilization. However, the specific contribution of each component to the reductive activation of CFoCF1 remains unclear. In this study, an in vitro assay system for evaluating thioredoxin-mediated CFoCF1 reduction is established, allowing manipulation of ΔµH+ components in isolated thylakoid membranes using specific chemicals. Our biochemical analyses revealed that ΔpH formation is essential for thioredoxin-mediated CFoCF1 reduction on the thylakoid membrane, whereas ΔΨ formation is nonessential.

Chemical genetic analysis of enoxolone inhibition of Clostridioides difficile toxin production reveals adenine deaminase and ATP synthase as antivirulence targets.

Toxins TcdA and TcdB are the main virulence factors of Clostridioides difficile, a leading cause of hospital-acquired diarrhea. Despite their importance, there is a significant knowledge gap of druggable targets for inhibiting toxin production. To address this, we screened nonantibiotic phytochemicals to identify potential chemical genetic probes to discover antivirulence drug targets. This led to the identification of 18ß-glycyrrhetinic acid (enoxolone), a licorice metabolite, as an inhibitor of TcdA and TcdB biosynthesis. Using affinity-based proteomics, potential targets were identified as ATP synthase subunit alpha (AtpA) and adenine deaminase (Ade, which catalyzes conversion of adenine to hypoxanthine in the purine salvage pathway). To validate these targets, a multifaceted approach was adopted. Gene silencing of ade and atpA inhibited toxin biosynthesis, while surface plasmon resonance and isothermal titration calorimetry molecular interaction analyses revealed direct binding of enoxolone to Ade. Metabolomics demonstrated enoxolone induced the accumulation of adenosine, while depleting hypoxanthine and ATP in C. difficile. Transcriptomics further revealed enoxolone dysregulated phosphate uptake genes, which correlated with reduced cellular phosphate levels. These findings suggest that enoxolone's cellular action is multitargeted. Accordingly, supplementation with both hypoxanthine and triethyl phosphate, a phosphate source, was required to fully restore toxin production in the presence of enoxolone. In conclusion, through the characterization of enoxolone, we identified promising antivirulence targets that interfere with nucleotide salvage and ATP synthesis, which may also block toxin biosynthesis.

Large-scale column-free purification of bovine F-ATP synthase.

Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers, tetramers, dimers, and monomers. In vitro investigations of mammalian F-ATP synthase are often limited by the ability to purify the oligomeric forms present in vivo at a quantity, stability, and purity that meets the demand of the planned experiment. We developed a purification approach for the isolation of bovine F-ATP synthase from heart muscle mitochondria that uses a combination of buffer conditions favoring inhibitor factor 1 binding and sucrose density gradient ultracentrifugation to yield stable complexes at high purity in the milligram range. By tuning the glyco-diosgenin to lauryl maltose neopentyl glycol ratio in a final gradient, fractions that are either enriched in tetrameric or monomeric F-ATP synthase can be obtained. It is expected that this large-scale column-free purification strategy broadens the spectrum of in vitro investigation on mammalian F-ATP synthase.

The inhibitor protein IF1 from mammalian mitochondria inhibits ATP hydrolysis but not ATP synthesis by the ATP synthase complex.

The hydrolytic activity of the ATP synthase in bovine mitochondria is inhibited by a protein called IF1, but bovine IF1 has no effect on the synthetic activity of the bovine enzyme in mitochondrial vesicles in the presence of a proton motive force. In contrast, it has been suggested based on indirect observations that human IFI inhibits both the hydrolytic and synthetic activities of the human ATP synthase and that the activity of human IF1 is regulated by the phosphorylation of Ser-14 of mature IF1. Here, we have made both human and bovine IF1 which are 81 and 84 amino acids long, respectively, and identical in 71.4% of their amino acids and have investigated their inhibitory effects on the hydrolytic and synthetic activities of ATP synthase in bovine submitochondrial particles. Over a wide range of conditions, including physiological conditions, both human and bovine IF1 are potent inhibitors of ATP hydrolysis, with no effect on ATP synthesis. Also, substitution of Ser-14 with phosphomimetic aspartic and glutamic acids had no effect on inhibitory properties, and Ser-14 is not conserved throughout mammals. Therefore, it is unlikely that the inhibitory activity of mammalian IF1 is regulated by phosphorylation of this residue.

A simple assay for inhibitors of mycobacterial oxidative phosphorylation.

Oxidative phosphorylation, the combined activities of the electron transport chain (ETC) and ATP synthase, has emerged as a valuable target for antibiotics to treat infection with Mycobacterium tuberculosis and related pathogens. In oxidative phosphorylation, the ETC establishes a transmembrane electrochemical proton gradient that powers ATP synthesis. Monitoring oxidative phosphorylation with luciferase-based detection of ATP synthesis or measurement of oxygen consumption can be technically challenging and expensive. These limitations reduce the utility of these methods for characterization of mycobacterial oxidative phosphorylation inhibitors. Here, we show that fluorescence-based measurement of acidification of inverted membrane vesicles (IMVs) can detect and distinguish between inhibition of the ETC, inhibition of ATP synthase, and nonspecific membrane uncoupling. In this assay, IMVs from Mycobacterium smegmatis are acidified either through the activity of the ETC or ATP synthase, the latter modified genetically to allow it to serve as an ATP-driven proton pump. Acidification is monitored by fluorescence from 9-amino-6-chloro-2-methoxyacridine, which accumulates and quenches in acidified IMVs. Nonspecific membrane uncouplers prevent both succinate- and ATP-driven IMV acidification. In contrast, the ETC Complex III2IV2 inhibitor telacebec (Q203) prevents succinate-driven acidification but not ATP-driven acidification, and the ATP synthase inhibitor bedaquiline prevents ATP-driven acidification but not succinate-driven acidification. We use the assay to show that, as proposed previously, lansoprazole sulfide is an inhibitor of Complex III2IV2, whereas thioridazine uncouples the mycobacterial membrane nonspecifically. Overall, the assay is simple, low cost, and scalable, which will make it useful for identifying and characterizing new mycobacterial oxidative phosphorylation inhibitors.

Cryo-EM reveals transition states of the Acinetobacter baumannii F1-ATPase rotary subunits γ and ε, unveiling novel compound targets.

Priority 1: critical WHO pathogen Acinetobacter baumannii depends on ATP synthesis and ATP:ADP homeostasis and its bifunctional F1FO-ATP synthase. While synthesizing ATP, it regulates ATP cleavage by its inhibitory ε subunit to prevent wasteful ATP consumption. We determined cryo-electron microscopy structures of the ATPase active A. baumannii F1-αßγεΔ134-139 mutant in four distinct conformational states, revealing four transition states and structural transformation of the ε's C-terminal domain, forming the switch of an ATP hydrolysis off- and an ATP synthesis on-state based. These alterations go in concert with altered motions and interactions in the catalytic- and rotary subunits of this engine. These A. baumannii interacting sites provide novel pathogen-specific targets for inhibitors, with the aim of ATP depletion and/or ATP synthesis and growth inhibition. Furthermore, the presented diversity to other bacterial F-ATP synthases extends the view of structural elements regulating such a catalyst.

Mitochondrial-derived vesicles retain membrane potential and contain a functional ATP synthase.

Vesicular transport is a means of communication. While cells can communicate with each other via secretion of extracellular vesicles, less is known regarding organelle-to organelle communication, particularly in the case of mitochondria. Mitochondria are responsible for the production of energy and for essential metabolic pathways in the cell, as well as fundamental processes such as apoptosis and aging. Here, we show that functional mitochondria isolated from Saccharomyces cerevisiae release vesicles, independent of the fission machinery. We isolate these mitochondrial-derived vesicles (MDVs) and find that they are relatively uniform in size, of about 100 nm, and carry selective protein cargo enriched for ATP synthase subunits. Remarkably, we further find that these MDVs harbor a functional ATP synthase complex. We demonstrate that these vesicles have a membrane potential, produce ATP, and seem to fuse with naive mitochondria. Our findings reveal a possible delivery mechanism of ATP-producing vesicles, which can potentially regenerate ATP-deficient mitochondria and may participate in organelle-to-organelle communication.

The consequence of ATP synthase dimer angle on mitochondrial morphology studied by cryo-electron tomography.

Mitochondrial ATP synthases form rows of dimers, which induce membrane curvature to give cristae their characteristic lamellar or tubular morphology. The angle formed between the central stalks of ATP synthase dimers varies between species. Using cryo-electron tomography and sub-tomogram averaging, we determined the structure of the ATP synthase dimer from the nematode worm C. elegans and show that the dimer angle differs from previously determined structures. The consequences of this species-specific difference at the dimer interface were investigated by comparing C. elegans and S. cerevisiae mitochondrial morphology. We reveal that C. elegans has a larger ATP synthase dimer angle with more lamellar (flatter) cristae when compared to yeast. The underlying cause of this difference was investigated by generating an atomic model of the C. elegans ATP synthase dimer by homology modelling. A comparison of our C. elegans model to an existing S. cerevisiae structure reveals the presence of extensions and rearrangements in C. elegans subunits associated with maintaining the dimer interface. We speculate that increasing dimer angles could provide an advantage for species that inhabit variable-oxygen environments by forming flatter more energetically efficient cristae.

H+-slip correlated to rotor free-wheeling as cause of F1FO-ATPase dysfunction in primary mitochondrial disorders.

Inborn errors of metabolism are related to mitochondrial disorders caused by dysfunction of the oxidative phosphorylation (OXPHOS) system. Congenital hypermetabolism in the infant is a rare disease belonging to Luft syndrome, nonthyroidal hypermetabolism, arising from a singular example of a defect in OXPHOS. The mitochondria lose coupling of mitochondrial substrates oxidation from the ADP phosphorylation. Since Luft syndrome is due to uncoupled cell respiration responsible for deficient in ATP production that originates in the respiratory complexes, a de novo heterozygous variant in the catalytic subunit of mitochondrial F1FO-ATPase arises as the main cause of an autosomal dominant syndrome of hypermetabolism associated with dysfunction in ATP production, which does not involve the respiratory complexes. The F1FO-ATPase works as an embedded molecular machine with a rotary action using two different motor engines. The FO, which is an integral domain in the membrane, dissipates the chemical potential difference for H+, a proton motive force (Δp), across the inner membrane to generate a torsion. The F1 domain-the hydrophilic portion responsible for ATP turnover-is powered by the molecular rotary action to synthesize ATP. The structural and functional coupling of F1 and FO domains support the energy transduction for ATP synthesis. The dissipation of Δp by means of an H+ slip correlated to rotor free-wheeling of the F1FO-ATPase has been discovered to cause enzyme dysfunction in primary mitochondrial disorders. In this insight, we try to offer commentary and analysis of the molecular mechanism in these impaired mitochondria.