Reversible binding of divalent cations to Ductin protein assemblies-A putative new regulatory mechanism of membrane traffic processes.

Ductins are a family of homologous and structurally similar membrane proteins with 2 or 4 trans-membrane alpha-helices. The active forms of the Ductins are membranous ring- or star-shaped oligomeric assemblies and they provide various pore, channel, gap-junction functions, assist in membrane fusion processes and also serve as the rotor c-ring domain of V-and F-ATPases. All functions of the Ductins have been reported to be sensitive to the presence of certain divalent metal cations (Me2+), most frequently Cu2+ or Ca2+ ions, for most of the better known members of the family, and the mechanism of this effect is not yet known. Given that we have earlier found a prominent Me2+ binding site in a well-characterised Ductin protein, we hypothesise that certain divalent cations can structurally modulate the various functions of Ductin assemblies via affecting their stability by reversible non-covalent binding to them. A fine control of the stability of the assembly ranging from separated monomers through a loosely/weakly to tightly/strongly assembled ring might render precise regulation of Ductin functions possible. The putative role of direct binding of Me2+ to the c-ring subunit of active ATP hydrolase in autophagy and the mechanism of Ca2+-dependent formation of the mitochondrial permeability transition pore are also discussed.

F-type proton-pumping ATPase mediates acid tolerance in Streptococcus mutans.

AIMS: Streptococcus mutans is highly sensitive to inhibitors of proton-pumping F-type ATPase (F-ATPase) under acidic conditions. Herein, we investigated the role of S. mutans F-ATPase in acid tolerance using a bacterium expressing the F-ATPase ß subunit at lower levels than the wild-type strain. METHODS AND RESULTS: We generated a mutant S. mutans expressing the catalytic ß subunit of F-ATPase at lower levels than the wild-type bacterium. The mutant cells exhibited a significantly slower growth rate at pH 5.30, whereas the rate was essentially the same as that of wild-type cells at pH 7.40. In addition, the colony-forming ability of the mutant was decreased at pH <4.30 but not at pH 7.40. Thus, the growth rate and survival of S. mutans expressing low levels of the ß subunit were reduced under acidic conditions. CONCLUSIONS: Together with our previous observations, this study indicates that F-ATPase is involved in the acid tolerance mechanism of S. mutans by secreting protons from the cytoplasm.

Proton Pumping ATPases: Rotational Catalysis, Physiological Roles in Oral Pathogenic Bacteria, and Inhibitors.

Proton pumping ATPases, both F-type and V/A-type ATPases, generate ATP using electrochemical energy or pump protons/sodium ions by hydrolyzing ATP. The enzymatic reaction and proton transport are coupled through subunit rotation, and this unique rotational mechanism (rotational catalysis) has been intensively studied. Single-molecule and thermodynamic analyses have revealed the detailed rotational mechanism, including the catalytically inhibited state and the roles of subunit interactions. In mammals, F- and V-ATPases are involved in ATP synthesis and organelle acidification, respectively. Most bacteria, including anaerobes, have F- and/or A-ATPases in the inner membrane. However, these ATPases are not believed to be essential in anaerobic bacteria since anaerobes generate sufficient ATP without oxidative phosphorylation. Recent studies suggest that F- and A-ATPases perform indispensable functions beyond ATP synthesis in oral pathogenic anaerobes; F-ATPase is involved in acid tolerance in Streptococcus mutans, and A-ATPase mediates nutrient import in Porphyromonas gingivalis. Consistently, inhibitors of oral bacterial F- and A-ATPases, such as phytopolyphenols and bedaquiline, strongly diminish growth and survival. Herein, we discuss rotational catalysis of bacterial F- and A-ATPases, and discuss their physiological roles, focusing on oral bacteria. We also review the effects of ATPase inhibitors on the growth and survival of oral pathogenic bacteria. The features of the catalytic mechanism and unique physiological roles in oral bacteria highlight the potential for proton pumping ATPases to serve as targets for oral antimicrobial agents.

Discovery and Study of Transmembrane Rotary Ion-Translocating Nano-Motors: F-ATPase/Synthase of Mitochondria/Bacteria and V-ATPase of Eukaryotic Cells.

This review discusses the history of discovery and study of the operation of the two rotary ion-translocating ATPase nano-motors: (i) F-ATPase/synthase (holocomplex F1FO) of mitochondria/bacteria and (ii) eukaryotic V-ATPase (holocomplex V1VO). Vacuolar adenosine triphosphatase (V-ATPase) is a transmembrane multisubunit complex found in all eukaryotes from yeast to humans. It is structurally and functionally similar to the F-ATPase/synthase of mitochondria/bacteria and the A-ATPase/synthase of archaebacteria, which indicates a common evolutionary origin of the rotary ion-translocating nano-motors built into cell membranes and invented by Nature billions of years ago. Previously we have published several reviews on this topic with appropriate citations of our original research. This review is focused on the historical analysis of the discovery and study of transmembrane rotary ion-translocating ATPase nano-motors functioning in bacteria, eukaryotic cells and mitochondria of animals.

Method to extract multiple states in F1-ATPase rotation experiments from jump distributions.

A method is proposed for analyzing fast (10 µs) single-molecule rotation trajectories in F1 adenosinetriphosphatase ([Formula: see text]-ATPase). This method is based on the distribution of jumps in the rotation angle that occur in the transitions during the steps between subsequent catalytic dwells. The method is complementary to the "stalling" technique devised by H. Noji et al. [Biophys. Rev. 9, 103-118, 2017], and can reveal multiple states not directly detectable as steps. A bimodal distribution of jumps is observed at certain angles, due to the system being in either of 2 states at the same rotation angle. In this method, a multistate theory is used that takes into account a viscoelastic fluctuation of the imaging probe. Using an established sequence of 3 specific states, a theoretical profile of angular jumps is predicted, without adjustable parameters, that agrees with experiment for most of the angular range. Agreement can be achieved at all angles by assuming a fourth state with an ∼10 µs lifetime and a dwell angle about 40° after the adenosine 5'-triphosphate (ATP) binding dwell. The latter result suggests that the ATP binding in one ß subunit and the adenosine 5'-diphosphate (ADP) release from another ß subunit occur via a transient whose lifetime is ∼10 µs and is about 6 orders of magnitude smaller than the lifetime for ADP release from a singly occupied [Formula: see text]-ATPase. An internal consistency test is given by comparing 2 independent ways of obtaining the relaxation time of the probe. They agree and are ∼15 µs.

Proton-pumping F-ATPase plays an important role in Streptococcus mutans under acidic conditions.

Streptococcus mutans, a bacterium mainly inhabiting the tooth surface, is a major pathogen of dental caries. The bacterium metabolizes sugars to produce acids, resulting in an acidic microenvironment in the dental plaque. Hence, S. mutans should possess a mechanism for surviving under acidic conditions. In the current study, we report the effects of inhibitors of Escherichia coli proton-pumping F-type ATPase (F-ATPase) on the activity of S. mutans enzyme, and the growth and survival of S. mutans under acidic conditions. Piceatannol, curcumin, and demethoxycurcumin strongly reduced the ATPase activity of S. mutans F-ATPase. Interestingly, these compounds inhibited the growth of S. mutans at pH 5.3 but not at pH 7.3. They also significantly reduced the colony-forming ability of S. mutans after incubation at pH 4.3, while showing essentially no effect at pH 7.3. These observations indicate that S. mutans is highly sensitive to F-ATPase inhibitors under acidic conditions and that F-ATPase plays an important role in acid tolerance of this bacterium.

Inhibition of F1 -ATPase from Trypanosoma brucei by its regulatory protein inhibitor TbIF1.

Hydrolysis of ATP by the mitochondrial F-ATPase is inhibited by a protein called IF1 . In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF1 , is expressed exclusively in the procyclic stage, where the F-ATPase is synthesizing ATP. In the bloodstream stage, where TbIF1 is absent, the F-ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF1 that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F1 -ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C-terminal portion of TbIF1 facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF1 , the inhibitory capacity of the C-terminally truncated TbIF1 diminishes with decreasing pH, similar to full length TbIF1 . This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C-terminally truncated TbIF1 form dimers and monomers, respectively. TbIF1 has no effect on bovine F1 -ATPase, and this difference in the mechanism of regulation of the F-ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases.

Theory of long binding events in single-molecule-controlled rotation experiments on F1-ATPase.

The theory of elastic group transfer for the binding and release rate constants for nucleotides in F1-ATPase as a function of the rotor angle is further extended in several respects. (i) A method is described for predicting the experimentally observed lifetime distribution of long binding events in the controlled rotation experiments by taking into account the hydrolysis and synthesis reactions occurring during these events. (ii) A method is also given for treating the long binding events in the experiments and obtaining the rate constants for the hydrolysis and synthesis reactions occurring during these events. (iii) The theory in the previous paper is given in a symmetric form, an extension that simplifies the application of the theory to experiments. It also includes a theory-based correction of the reported "on" and "off" rates by calculating the missed events. A near symmetry of the data about the angle of -40° and a "turnover" in the binding rate data vs. rotor angle for angles greater than [Formula: see text]40° is also discussed.

Operating principles of rotary molecular motors: differences between F1 and V1 motors.

Among the many types of bioenergy-transducing machineries, F- and V-ATPases are unique bio- and nano-molecular rotary motors. The rotational catalysis of F1-ATPase has been investigated in detail, and molecular mechanisms have been proposed based on the crystal structures of the complex and on extensive single-molecule rotational observations. Recently, we obtained crystal structures of bacterial V1-ATPase (A3B3 and A3B3DF complexes) in the presence and absence of nucleotides. Based on these new structures, we present a novel model for the rotational catalysis mechanism of V1-ATPase, which is different from that of F1-ATPases.

On the structural possibility of pore-forming mitochondrial FoF1 ATP synthase.

The mitochondrial permeability transition is an inner mitochondrial membrane event involving the opening of the permeability transition pore concomitant with a sudden efflux of matrix solutes and breakdown of membrane potential. The mitochondrial F(o)F(1) ATP synthase has been proposed as the molecular identity of the permeability transition pore. The likeliness of potential pore-forming sites in the mitochondrial F(o)F(1) ATP synthase is discussed and a new model, the death finger model, is described. In this model, movement of a p-side density that connects the lipid-plug of the c-ring with the distal membrane bending Fo domain allows reversible opening of the c-ring and structural cross-talk with OSCP and the catalytic (αß)(3) hexamer. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

ATP synthase from Escherichia coli: Mechanism of rotational catalysis, and inhibition with the ε subunit and phytopolyphenols.

ATP synthases (FoF1) are found ubiquitously in energy-transducing membranes of bacteria, mitochondria, and chloroplasts. These enzymes couple proton transport and ATP synthesis or hydrolysis through subunit rotation, which has been studied mainly by observing single molecules. In this review, we discuss the mechanism of rotational catalysis of ATP synthases, mainly that from Escherichia coli, emphasizing the high-speed and stochastic rotation including variable rates and an inhibited state. Single molecule studies combined with structural information of the bovine mitochondrial enzyme and mutational analysis have been informative as to an understanding of the catalytic site and the interaction between rotor and stator subunits. We discuss the similarity and difference in structure and inhibitory regulation of F1 from bovine and E. coli. Unlike the crystal structure of bovine F1 (α3ß3γ), that of E. coli contains a ε subunit, which is a known inhibitor of bacterial and chloroplast F1 ATPases. The carboxyl terminal domain of E. coli ε (εCTD) interacts with the catalytic and rotor subunits (ß and γ, respectively), and then inhibits rotation. The effects of phytopolyphenols on F1-ATPase are also discussed: one of them, piceatannol, lowered the rotational speed by affecting rotor/stator interactions.

The pH-dependent effects of combining ethanol with fluoride on proton permeability in Streptococcus mutans / 대한구강보건학회지

OBJECTIVES: The aim of this research was to determine the pH-dependent changes in F-ATPase activity and proton fluxes in Streptococcus mutans (S. mutans) as induced by varying the concentration of fluoride ±10 mM (0.058% (v/v)) ethanol. METHODS: S. mutans UA159 was grown in Brain Heart Infusion medium at pH 4.8, 6.8, or 8.8. The F-ATPase assay was initiated by the addition of ATP, and stopped by adding 10% trichloroacetic acid. For the proton flux assay, bacterial suspensions were titrated to pH 4.6 with 0.5 M HCl, and then 0.5 M HCl was added to decrease the pH values in units of approximately 0.4 pH. The subsequent increase in pH was monitored using a glass electrode. To disrupt the cell membrane, 10% (v/v) butanol was added to the suspensions after 80 minutes. RESULTS: At all pH levels, fluoride ±10 mM ethanol not only decreased F-ATPase activity but also increased the proton permeability of S. mutans. The largest effects were observed at pH 4.8. Ethanol enhanced these effects only at pH 4.8. CONCLUSIONS: A very low concentration of ethanol enhanced the action of fluoride on F-ATPase activity and the proton permeability in S. mutans at acidic pH levels. We expect that low concentrations of ethanol may be used together with fluoride and/or other anticaries agents to develop more effective anticaries preparations.

Purification, characterization and crystallization of the F-ATPase from Paracoccus denitrificans.

The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F1-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex.

Structure of a catalytic dimer of the α- and ß-subunits of the F-ATPase from Paracoccus denitrificans at 2.3†Šresolution.

The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied. Paracoccus denitrificans is a member of the α-proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. The P. denitrificans F-ATPase is an example of a eubacterial F-ATPase that can carry out ATP synthesis only, whereas many others can catalyse both the synthesis and the hydrolysis of ATP. Inhibition of the ATP hydrolytic activity of the P. denitrificans F-ATPase involves the ζ inhibitor protein, an α-helical protein that binds to the catalytic F1 domain of the enzyme. This domain is a complex of three α-subunits and three ß-subunits, and one copy of each of the γ-, δ- and ℇ-subunits. Attempts to crystallize the F1-ζ inhibitor complex yielded crystals of a subcomplex of the catalytic domain containing the α- and ß-subunits only. Its structure was determined to 2.3 Šresolution and consists of a heterodimer of one α-subunit and one ß-subunit. It has no bound nucleotides, and it corresponds to the `open' or `empty' catalytic interface found in other F-ATPases. The main significance of this structure is that it aids in the determination of the structure of the intact membrane-bound F-ATPase, which has been crystallized.

Strong inhibitory effects of curcumin and its demethoxy analog on Escherichia coli ATP synthase F1 sector.

Curcumin, a dietary phytopolyphenol isolated from a perennial herb (Curcuma longa), is a well-known compound effective for bacterial infections and tumors, and also as an antioxidant. In this study, we report the inhibitory effects of curcumin and its analogs on the Escherichia coli ATP synthase F1 sector. A structure-activity relationship study indicated the importance of 4'-hydroxy groups and a ß-diketone moiety for the inhibition. The 3'-demethoxy analog (DMC) inhibited F1 more strongly than curcumin did. Furthermore, these compounds inhibited E. coli growth through oxidative phosphorylation, consistent with their effects on ATPase activity. These results suggest that the two compounds affected bacterial growth through inhibition of ATP synthase. Derivatives including bis(arylmethylidene)acetones (C5 curcuminoids) exhibited only weak activity toward ATPase and bacterial growth.

Individual interactions of the b subunits within the stator of the Escherichia coli ATP synthase.

FOF1 ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of FOF1. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-ß, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. bI and bII). The results show the b dimer to be located at a non-catalytic α/ß cleft, with bI close to subunit α, whereas bII is proximal to subunit ß. Furthermore, bI can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-bI-α, bII-ß, α-bI-bII-ß, and a-bI-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that bI is functionally related to the single b subunit present in mitochondrial ATP synthase.

F-ATPase subunit uncEBF mRNA expression in Streptococcus mutans clinical isolates / 实用口腔医学杂志

Objective:To investigate the gene expression variety of different genotype of F-ATPase subunit uncEBF in Streptococcus mutans (S.mutans) and to evaluate the relationship among uncEBF gene expression levels, genotypes and the acidurance ability of S.mutans. Methods:The relative expression quantity of uncEBF gene against the housekeeping gene recA was determined by the two-step method of semi-quantitative RT-PCR in 18 clinical isolates of S.mutans(7 with genotype A uncEBF and 11 with genotyp B,10 with high acidurance and 8 with low). A gel documentation system and QUANTITY ONES software were used to assay the data. Results:uncEBF mRNA expression level in the isolates with genotype A uncEBF was higher than that in those with genotype B(P