Ablation of Fatty Acid Transport Protein-4 Enhances Cone Survival, M-cone Vision, and Synthesis of Cone-Tropic 9-cis-Retinal in rd12 Mouse Model of Leber Congenital Amaurosis.

The canonical visual cycle employing RPE65 as the retinoid isomerase regenerates 11-cis-retinal to support both rod- and cone-mediated vision. Mutations of RPE65 are associated with Leber congenital amaurosis that results in rod and cone photoreceptor degeneration and vision loss of affected patients at an early age. Dark-reared Rpe65-/- mouse has been known to form isorhodopsin that employs 9-cis-retinal as the photosensitive chromophore. The mechanism regulating 9-cis-retinal synthesis and the role of the endogenous 9-cis-retinal in cone survival and function remain largely unknown. In this study, we found that ablation of fatty acid transport protein-4 (FATP4), a negative regulator of 11-cis-retinol synthesis catalyzed by RPE65, increased the formation of 9-cis-retinal, but not 11-cis-retinal, in a light-independent mechanism in both sexes of RPE65-null rd12 mice. Both rd12 and rd12;Fatp4-/- mice contained a massive amount of all-trans-retinyl esters in the eyes, exhibiting comparable scotopic vision and rod degeneration. However, expression levels of M- and S-opsins as well as numbers of M- and S-cones surviving in the superior retinas of rd12;Fatp4-/ - mice were at least twofold greater than those in age-matched rd12 mice. Moreover, FATP4 deficiency significantly shortened photopic b-wave implicit time, improved M-cone visual function, and substantially deaccelerated the progression of cone degeneration in rd12 mice, whereas FATP4 deficiency in mice with wild-type Rpe65 alleles neither induced 9-cis-retinal formation nor influenced cone survival and function. These results identify FATP4 as a new regulator of synthesis of 9-cis-retinal, which is a "cone-tropic" chromophore supporting cone survival and function in the retinas with defective RPE65.

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.

Bladder-cancer-derived exosomal circRNA_0013936 promotes suppressive immunity by up-regulating fatty acid transporter protein 2 and down-regulating receptor-interacting protein kinase 3 in PMN-MDSCs.

BACKGROUND: Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) is one of the causes of tumor immune tolerance and failure of cancer immunotherapy. Here, we found that bladder cancer (BCa)-derived exosomal circRNA_0013936 could enhance the immunosuppressive activity of PMN-MDSCs by regulating the expression of fatty acid transporter protein 2 (FATP2) and receptor-interacting protein kinase 3 (RIPK3). However, the underlying mechanism remains largely unknown. METHODS: BCa-derived exosomes was isolated and used for a series of experiments. RNA sequencing was used to identify the differentially expressed circRNAs. Western blotting, immunohistochemistry, immunofluorescence, qRT-PCR, ELISA and Flow cytometry were performed to reveal the potential mechanism of circRNA_0013936 promoting the immunosuppressive activity of PMN-MDSC. RESULTS: CircRNA_0013936 enriched in BCa-derived exosomes could promote the expression of FATP2 and inhibit the expression of RIPK3 in PMN-MDSCs. Mechanistically, circRNA_0013936 promoted the expression of FATP2 and inhibited the expression of RIPK3 expression via sponging miR-320a and miR-301b, which directly targeted JAK2 and CREB1 respectively. Ultimately, circRNA_0013936 significantly inhibited the functions of CD8+ T cells by up-regulating FATP2 through the circRNA_0013936/miR-320a/JAK2 pathway, and down-regulating RIPK3 through the circRNA_0013936/miR-301b/CREB1 pathway in PMN-MDSCs. CONCLUSIONS: BCa-derived exosomal circRNA_0013936 promotes suppressive immunity by up-regulating FATP2 through the circRNA_0013936/miR-320a/JAK2 pathway and down-regulating RIPK3 through the circRNA_0013936/miR-301b-3p/CREB1 pathway in PMN-MDSCs. These findings help to find new targets for clinical treatment of human bladder cancer.

Variations of the Mycobacterium abscessus F-ATP synthase subunit a-c interface alter binding and potency of the anti-TB drug bedaquiline.

The anti-tuberculosis therapeutic bedaquiline (BDQ) is used against Mycobacterium abscessus. In M. abscessus BDQ is only bacteriostatic and less potent compared to M. tuberculosis or M. smegmatis. Here we demonstrate its reduced ATP synthesis inhibition against M. abscessus inside-out vesicles, including the F1FO-ATP synthase. Molecular dynamics simulations and binding free energy calculations highlight the differences in drug-binding of the M. abscessus and M. smegmatis FO-domain at the lagging site, where the drug deploys its mechanistic action, inhibiting ATP synthesis. These data pave the way for improved anti-M. abscessus BDQ analogs.

Atomic insights of an up and down conformation of the Acinetobacter baumannii F1 -ATPase subunit ε and deciphering the residues critical for ATP hydrolysis inhibition and ATP synthesis.

The Acinetobacter baumannii F1 FO -ATP synthase (α3 :ß3 :γ:δ:ε:a:b2 :c10 ), which is essential for this strictly respiratory opportunistic human pathogen, is incapable of ATP-driven proton translocation due to its latent ATPase activity. Here, we generated and purified the first recombinant A. baumannii F1 -ATPase (AbF1 -ATPase) composed of subunits α3 :ß3 :γ:ε, showing latent ATP hydrolysis. A 3.0 Å cryo-electron microscopy structure visualizes the architecture and regulatory element of this enzyme, in which the C-terminal domain of subunit ε (Abε) is present in an extended position. An ε-free AbF1 -ɑßγ complex generated showed a 21.5-fold ATP hydrolysis increase, demonstrating that Abε is the major regulator of AbF1 -ATPase's latent ATP hydrolysis. The recombinant system enabled mutational studies of single amino acid substitutions within Abε or its interacting subunits ß and γ, respectively, as well as C-terminal truncated mutants of Abε, providing a detailed picture of Abε's main element for the self-inhibition mechanism of ATP hydrolysis. Using a heterologous expression system, the importance of Abε's C-terminus in ATP synthesis of inverted membrane vesicles, including AbF1 FO -ATP synthases, has been explored. In addition, we are presenting the first NMR solution structure of the compact form of Abε, revealing interaction of its N-terminal ß-barrel and C-terminal ɑ-hairpin domain. A double mutant of Abε highlights critical residues for Abε's domain-domain formation which is important also for AbF1 -ATPase's stability. Abε does not bind MgATP, which is described to regulate the up and down movements in other bacterial counterparts. The data are compared to regulatory elements of F1 -ATPases in bacteria, chloroplasts, and mitochondria to prevent wasting of ATP.

F-ATP synthase inhibitory factor 1 and mitochondria-organelle interactions: New insight and implications.

Mitochondria are metabolic hub, and act as primary sites for reactive oxygen species (ROS) and metabolites generation. Mitochondrial Ca2+ uptake contributes to Ca2+ storage. Mitochondria-organelle interactions are important for cellular metabolic adaptation, biosynthesis, redox balance, cell fate. Organelle communications are mediated by Ca2+/ROS signals, vesicle transport and membrane contact sites. The permeability transition pore (PTP) is an unselective channel that provides a release pathway for Ca2+/ROS, mtDNA and metabolites. F-ATP synthase inhibitory factor 1 (IF1) participates in regulation of PTP opening and is required for the translocation of transcriptional factors c-Myc/PGC1α to mitochondria to stimulate metabolic switch. IF1, a mitochondrial specific protein, has been suggested to regulate other organelles including nucleus, endoplasmic reticulum and lysosomes. IF1 may be able to mediate mitochondria-organelle interactions and cellular physiology through regulation of PTP activity.

Ligand-free mitochondria-localized mutant AR-induced cytotoxicity in spinal bulbar muscular atrophy.

Spinal bulbar muscular atrophy (SBMA), the first identified CAG-repeat expansion disorder, is an X-linked neuromuscular disorder involving CAG-repeat-expansion mutations in the androgen receptor (AR) gene. We utilized CRISPR-Cas9 gene editing to engineer novel isogenic human induced pluripotent stem cell (hiPSC) models, consisting of isogenic AR knockout, control and disease lines expressing mutant AR with distinct repeat lengths, as well as control and disease lines expressing FLAG-tagged wild-type and mutant AR, respectively. Adapting a small-molecule cocktail-directed approach, we differentiate the isogenic hiPSC models into motor neuron-like cells with a highly enriched population to uncover cell-type-specific mechanisms underlying SBMA and to distinguish gain- from loss-of-function properties of mutant AR in disease motor neurons. We demonstrate that ligand-free mutant AR causes drastic mitochondrial dysfunction in neurites of differentiated disease motor neurons due to gain-of-function mechanisms and such cytotoxicity can be amplified upon ligand (androgens) treatment. We further show that aberrant interaction between ligand-free, mitochondria-localized mutant AR and F-ATP synthase is associated with compromised mitochondrial respiration and multiple other mitochondrial impairments. These findings counter the established notion that androgens are requisite for mutant AR-induced cytotoxicity in SBMA, reveal a compelling mechanistic link between ligand-free mutant AR, F-ATP synthase and mitochondrial dysfunction, and provide innovative insights into motor neuron-specific therapeutic interventions for SBMA.

N-Acyl phenothiazines as mycobacterial ATP synthase inhibitors: Rational design, synthesis and in vitro evaluation against drug sensitive, RR and MDR-TB.

The mycobacterial F-ATP synthase is responsible for the optimal growth, metabolism and viability of Mycobacteria, establishing it as a validated target for the development of anti-TB therapeutics. Herein, we report the discovery of an N-acyl phenothiazine derivative, termed PT6, targeting the mycobacterial F-ATP synthase. PT6 is bactericidal and active against the drug sensitive, Rifampicin-resistant as well as Multidrug-resistant tuberculosis strains. Compound PT6 showed noteworthy inhibition of F-ATP synthesis, exhibiting an IC50 of 0.788 µM in M. smegmatis IMVs and was observed that it could deplete intracellular ATP levels, exhibiting an IC50 of 30 µM. PT6 displayed a high selectivity towards mycobacterial ATP synthase compared to mitochondrial ATP synthase. Compound PT6 showed a minor synergistic effect in combination with Rifampicin and Isoniazid. PT6 demonstrated null cytotoxicity as confirmed by assessing its toxicity against VERO cell lines. Further, the binding mechanism and the activity profile of PT6 were validated by employing in silico techniques such as molecular docking, Prime MM/GBSA, DFT and ADMET analysis. These results suggest that PT6 presents an attractive lead for the discovery of a novel class of mycobacterial F-ATP synthase inhibitors.

Mitochondrial ATP synthase inhibitory factor 1 interacts with the p53-cyclophilin D complex and promotes opening of the permeability transition pore.

The mitochondrial permeability transition pore (PTP) is a Ca2+-dependent megachannel that plays an important role in mitochondrial physiology and cell fate. Cyclophilin D (CyPD) is a well-characterized PTP regulator, and its binding to the PTP favors pore opening. It has previously been shown that p53 physically interacts with CyPD and opens the PTP during necrosis. Accumulating studies also suggest that the F-ATP synthase contributes to the regulation and formation of the PTP. F-ATP synthase IF1 (mitochondrial ATP synthase inhibitory factor 1) is a natural inhibitor of F-ATP synthase activity; however, whether IF1 participates in the modulation of PTP opening is basically unknown. Here, we demonstrate using calcium retention capacity assay that IF1 overexpression promotes mitochondrial permeability transition via opening of the PTP. Intriguingly, we show that IF1 can interact with the p53-CyPD complex and facilitate cell death. We also demonstrate that the presence of IF1 is necessary for the formation of p53-CyPD complex. Therefore, we suggest that IF1 regulates the PTP via interaction with the p53-CyPD complex, and that IF1 is necessary for the inducing effect of p53-CyPD complex on PTP opening.

Study protocol of OmegaROP-2 prospective study: expression of placental fatty acid receptors in preterm newborns with retinopathy of prematurity.

BACKGROUND: Incomplete vascularization of the retina in preterm infants carries a risk of retinopathy of prematurity (ROP). Progress in neonatal resuscitation in developing countries has led to the survival of an increasing number of premature infants, resulting in an increased rate of ROP and consequently in visual disability. Strategies to reduce ROP involve optimizing oxygen saturation, nutrition, and normalizing factors such as insulin-like growth factor 1 and n-3 long-chain polyunsaturated fatty acids (LC-PUFA). Our previous study, OmegaROP, showed that there is an accumulation or retention of docosahexaenoic acid (DHA) in mothers of infants developing ROP, suggesting abnormalities in the LC-PUFA placental transfer via fatty acid transporting proteins. The present study aims to better understand the LC-PUFA transport dysfunction in the fetoplacental unit during pregnancy and to find a novel target for the prevention of ROP development. METHODS: The study protocol is designed to evaluate the correlation between the expression level of placental fatty acid receptors and ROP occurrence. This ongoing study will include 100 mother-infant dyads: mother-infant dyads born before 29 weeks of gestational age (GA) and mother-infant dyads with full-term pregnancies. Recruitment is planned over a period of 46 months. Maternal and cord blood samples as well as placental tissue samples will be taken following delivery. ROP screening will be performed using wide-field camera imaging according to the International Classification of ROP consensus statement. DISCUSSION: The results of this study will have a tangible impact on public health. Indeed, if we show a correlation between the expression level of placental omega-3 receptors and the occurrence of ROP, it would be an essential step in discovering novel pathophysiological mechanisms involved in this retinopathy. TRIAL REGISTRATION: NCT04819893.

Structural Elements Involved in ATP Hydrolysis Inhibition and ATP Synthesis of Tuberculosis and Nontuberculous Mycobacterial F-ATP Synthase Decipher New Targets for Inhibitors.

The F1FO-ATP synthase is required for the viability of tuberculosis (TB) and nontuberculous mycobacteria (NTM) and has been validated as a drug target. Here, we present the cryo-EM structures of the Mycobacterium smegmatis F1-ATPase and the F1FO-ATP synthase with different nucleotide occupation within the catalytic sites and visualize critical elements for latent ATP hydrolysis and efficient ATP synthesis. Mutational studies reveal that the extended C-terminal domain (αCTD) of subunit α is the main element for the self-inhibition mechanism of ATP hydrolysis for TB and NTM bacteria. Rotational studies indicate that the transition between the inhibition state by the αCTD and the active state is a rapid process. We demonstrate that the unique mycobacterial γ-loop and subunit δ are critical elements required for ATP formation. The data underline that these mycobacterium-specific elements of α, γ, and δ are attractive targets, providing a platform for the discovery of species-specific inhibitors.

Anti-Mycobacterium abscessus Activity of Tuberculosis F-ATP Synthase Inhibitor GaMF1.

New drug targets and molecules with bactericidal activity are needed against the respiratory mycobacterial pathogen Mycobacterium abscessus. Employing a lead repurposing strategy, the antituberculosis compound GaMF1 was tested against M. abscessus. Whole-cell and ATP synthesis assays demonstrated that GaMF1 inhibits growth and kills M. abscessus by targeting the F-ATP synthase. GaMF1's anti-M. abscessus activity increased in combination with clofazimine, rifabutin, or amikacin. The study expands the repertoire of anti-M. abscessus compounds targeting oxidative phosphorylation.

Deletion of fatty acid transport protein 2 (FATP2) in the mouse liver changes the metabolic landscape by increasing the expression of PPARα-regulated genes.

Fatty acid transport protein 2 (FATP2) is highly expressed in the liver, small intestine, and kidney, where it functions in both the transport of exogenous long-chain fatty acids and the activation of very-long-chain fatty acids. Here, using a murine model, we investigated the phenotypic impacts of deleting FATP2, followed by a transcriptomic analysis using unbiased RNA-Seq to identify concomitant changes in the liver transcriptome. WT and FATP2-null (Fatp2-/-) mice (5 weeks) were maintained on a standard chow diet for 6 weeks. The Fatp2-/- mice had reduced weight gain, lowered serum triglyceride, and increased serum cholesterol levels and attenuated dietary fatty acid absorption. Transcriptomic analysis of the liver revealed 258 differentially expressed genes in male Fatp2-/- mice and a total of 91 in female Fatp2-/- mice. These genes mapped to the following gene ontology categories: fatty acid degradation, peroxisome biogenesis, fatty acid synthesis, and retinol and arachidonic acid metabolism. Targeted RT-quantitative PCR verified the altered expression of selected genes. Of note, most of the genes with increased expression were known to be regulated by peroxisome proliferator-activated receptor α (PPARα), suggesting that FATP2 activity is linked to a PPARα-specific proximal ligand. Targeted metabolomic experiments in the Fatp2-/- liver revealed increases of total C16:0, C16:1, and C18:1 fatty acids; increases in lipoxin A4 and prostaglandin J2; and a decrease in 20-hydroxyeicosatetraenoic acid. We conclude that the expression of FATP2 in the liver broadly affects the metabolic landscape through PPARα, indicating that FATP2 provides an important role in liver lipid metabolism through its transport or activation activities.

Regulation of ROS in myeloid-derived suppressor cells through targeting fatty acid transport protein 2 enhanced anti-PD-L1 tumor immunotherapy.

Despite the remarkable success and efficacy of immune checkpoint blockade (ICB) therapy against the PD-1/PD-L1 axis, it induces sustained responses in a sizeable minority of cancer patients due to the activation of immunosuppressive factors such as myeloid-derived suppressor cells (MDSCs). Inhibiting the immunosuppressive function of MDSCs is critical for successful cancer ICB therapy. Interestingly, lipid metabolism is a crucial factor in modulating MDSCs function. Fatty acid transport protein 2 (FATP2) conferred the function of PMN-MDSCs in cancer via the upregulation of arachidonic acid metabolism. However, whether regulating lipid accumulation in MDSCs by targeting FATP2 could block MDSCs reactive oxygen species (ROS) production and enhance PD-L1 blockade-mediated tumor immunotherapy remains unexplored. Here we report that FATP2 regulated lipid accumulation, ROS, and immunosuppressive function of MDSCs in tumor-bearing mice. Tumor cells-derived granulocyte macrophage-colony stimulating factor (GM-CSF) induced FATP2 expression in MDSCs by activation of STAT3 signaling pathway. Pharmaceutical blockade of FATP2 expression in MDSCs by lipofermata decreased lipid accumulation, reduced ROS, blocked immunosuppressive activity, and consequently inhibited tumor growth. More importantly, lipofermata inhibition of FATP2 in MDSCs enhanced anti-PD-L1 tumor immunotherapy via the upregulation of CD107a and reduced PD-L1 expression on tumor-infiltrating CD8+T-cells. Furthermore, the combination therapy blocked MDSC's suppressive role on T- cells thereby enhanced T-cell's ability for the production of IFN-γ. These findings indicate that FATP2 plays a key role in modulating lipid accumulation-induced ROS in MDSCs and targeting FATP2 in MDSCs provides a novel therapeutic approach to enhance anti-PD-L1 cancer immunotherapy.

Requirement for the intestinal epithelial insulin-like growth factor-1 receptor in the intestinal responses to glucagon-like peptide-2 and dietary fat.

The intestinal hormone, glucagon-like peptide-2 (GLP-2), enhances the enterocyte chylomicron production. However, GLP-2 is known to require the intestinal-epithelial insulin-like growth factor-1 receptor (IE-IGF-1R) for its other actions to increase intestinal growth and barrier function. The role of the IE-IGF-1R in enterocyte lipid handling was thus tested in the GLP-2 signaling pathway, as well as in response to a Western diet (WD). IE-IGF-1R knockout (KO) and control mice were treated for 11 days with h(GLY2 )GLP-2 or fed a WD for 18 weeks followed by a duodenal fat tolerance test with C14 -labeled triolein. Human Caco-2BBE cells were treated with an IGF-1R antagonist or signaling inhibitors to determine triglyceride-associated protein expression. The IE-IGF-1R was required for GLP-2-induced increases in CD36 and FATP-4 in chow-fed mice, and for expression in vitro; FATP-4 also required PI3K/Akt. Although WD-fed IE-IGF-1R KO mice demonstrated normal CD36 expression, the protein was incorrectly localized 2h post-duodenal fat administration. IE-IGF-1R KO also prevented the WD-induced increase in MTP and decrease in APOC3, increased jejunal mucosal C14 -fat accumulation, and elevated plasma triglyceride and C14 -fat levels. Collectively, these studies elucidate new roles for the IE-IGF-1R in enterocyte lipid handling, under basal conditions and in response to GLP-2 and WD-feeding.

The role of FATP1 in lipid accumulation: a review.

Lipid accumulation in mammals has been widely studied for decades due to its significant association with obesity in humans and meat quality in livestock animals. Fatty acid transport 1 (FATP1) is an evolutionarily conserved protein that localizes to the plasma membrane to enhance the transportation of fatty acids (FAs). In line with this function, FATP1 is involved in the metabolism of FAs, including their esterification and oxidation. In addition, the expression of FATP1 can be regulated by several energy-related factors, such as insulin and PPAR activators and transcription factors. These events connect FATP1 with cellular lipid accumulation. Recently, several studies have suggested that FATP1 acts as a facilitator in cellular lipid accumulation, whereas others hold a contrary view. Here, we will review these data and probe the possibility that FATP1 acts as a regulator in lipid accumulation, which will provide effective information for studies on the relationship between FATP1 and obesity in humans and meat quality in livestock animals.

Modulation and Pharmacology of the Mitochondrial Permeability Transition: A Journey from F-ATP Synthase to ANT.

The permeability transition (PT) is an increased permeation of the inner mitochondrial membrane due to the opening of the PT pore (PTP), a Ca2+-activated high conductance channel involved in Ca2+ homeostasis and cell death. Alterations of the PTP have been associated with many pathological conditions and its targeting represents an incessant challenge in the field. Although the modulation of the PTP has been extensively explored, the lack of a clear picture of its molecular nature increases the degree of complexity for any target-based approach. Recent advances suggest the existence of at least two mitochondrial permeability pathways mediated by the F-ATP synthase and the ANT, although the exact molecular mechanism leading to channel formation remains elusive for both. A full comprehension of this to-pore conversion will help to assist in drug design and to develop pharmacological treatments for a fine-tuned PT regulation. Here, we will focus on regulatory mechanisms that impinge on the PTP and discuss the relevant literature of PTP targeting compounds with particular attention to F-ATP synthase and ANT.

The Unique C-Terminal Extension of Mycobacterial F-ATP Synthase Subunit α Is the Major Contributor to Its Latent ATP Hydrolysis Activity.

Mycobacterial F1Fo-ATP synthases (α3:ß3:γ:δ:ε:a:b:b':c9 ) are incapable of ATP-driven proton translocation due to their latent ATPase activity. This prevents wasting of ATP and altering of the proton motive force, whose dissipation is lethal to mycobacteria. We demonstrate that the mycobacterial C-terminal extension of nucleotide-binding subunit α contributes mainly to the suppression of ATPase activity in the recombinant mycobacterial F1-ATPase. Using C-terminal deletion mutants, the regions responsible for the enzyme's latency were mapped, providing a new compound epitope.

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

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

3D reconstruction and flexibility of the hybrid engine Acetobacterium woodii F-ATP synthase.

The Na+-translocating F1FO ATP synthase from Acetobacterium woodii (AwF-ATP synthase) with a subunit stoichiometry of α3:ß3:γ:δ:ε:a:b2:(c2/3)9:c1 represents an evolutionary path between ATP-synthases and vacuolar ATPases, by containing a heteromeric rotor c-ring, composed of subunits c1, c2 and c3, and an extra loop (γ195-211) within the rotary γ subunit. Here, the recombinant AwF-ATP synthase was subjected to negative stain electron microscopy and single particle analysis. The reference free 2D class averages revealed high flexibility of the enzyme, wherein the F1 and FO domains distinctively bended to adopt multiple conformations. Moreover, both the F1 and FO domains tilted relative to each other to a maximum extent of 28° and 30°, respectively. The first 3D reconstruction of the AwF-ATP synthase was determined which accommodates well the modelled structure of the AwF-ATP synthase as well as the γ195-211-loop. Molecular simulations of the enzyme underlined the bending features and flexibility observed in the electron micrographs, and enabled assessment of the dynamics of the extra γ195-211-loop.