Regulatory Mechanisms and Environmental Adaptation of the F-ATPase Family.

The F-type ATPase family of enzymes, including ATP synthases, are found ubiquitously in biological membranes. ATP synthesis from ADP and inorganic phosphate is driven by an electrochemical H+ gradient or H+ motive force, in which intramolecular rotation of F-type ATPase is generated with H+ transport across the membranes. Because this rotation is essential for energy coupling between catalysis and H+-transport, regulation of the rotation is important to adapt to environmental changes and maintain ATP concentration. Recently, a series of cryo-electron microscopy images provided detailed insights into the structure of the H+ pathway and the multiple subunit arrangement. However, the regulatory mechanism of the rotation has not been clarified. This review describes the inhibition mechanism of ATP hydrolysis in bacterial enzymes. In addition, properties of the F-type ATPase of Streptococcus mutans, which acts as a H+-pump in an acidic environment, are described. These findings may help in the development of novel antimicrobial agents.

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.

The carboxyl-terminal helical domain of the ATP synthase γ subunit is involved in ε subunit conformation and energy coupling.

The γ subunit located at the center of ATP synthase (FOF1) plays critical roles in catalysis. Escherichia coli mutant with Pro substitution of the γ subunit residue γLeu218, which are located the rotor shaft near the c subunit ring, decreased NADH-driven ATP synthesis activity and ATP hydrolysis-dependent H+ transport of membranes to ~60% and ~40% of the wild type, respectively, without affecting FOF1 assembly. Consistently, the mutant was defective in growth by oxidative phosphorylation, indicating that energy coupling is impaired by the mutation. The ε subunit conformations in the γLeu218Pro mutant enzyme were investigated by cross-linking between cysteine residues introduced into both the ε subunit (εCys118 and εCys134, in the second helix and the hook segment, respectively) and the γ subunit (γCys99 and γCys260, located in the globular domain and the carboxyl-terminal helix, respectively). In the presence of ADP, the two γ260 and ε134 cysteine residues formed a disulfide bond in both the γLeu218Pro mutant and the wild type, indicating that the hook segment of ε subunit penetrates into the α3ß3-ring along with the γ subunits in both enzymes. However, γ260/ε134 cross-linking in the γLeu218Pro mutant decreased significantly in the presence of ATP, whereas this effect was small in the wild type. These results suggested that the γ subunit carboxyl-terminal helix containing γLeu218 is involved in the conformation of the ε subunit hook region during ATP hydrolysis and, therefore, is required for energy coupling in FOF1.

A unique mechanism of curcumin inhibition on F1 ATPase.

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

Elastic rotation of Escherichia coli F(O)F(1) having ε subunit fused with cytochrome b(562) or flavodoxin reductase.

Intra-molecular rotation of FOF1 ATP synthase enables cooperative synthesis and hydrolysis of ATP. In this study, using a small gold bead probe, we observed fast rotation close to the real rate that would be exhibited without probes. Using this experimental system, we tested the rotation of FOF1 with the ε subunit connected to a globular protein [cytochrome b562 (ε-Cyt) or flavodoxin reductase (ε-FlavR)], which is apparently larger than the space between the central and the peripheral stalks. The enzymes containing ε-Cyt and ε-FlavR showed continual rotations with average rates of 185 and 148 rps, respectively, similar to the wild type (172 rps). However, the enzymes with ε-Cyt or ε-FlavR showed a reduced proton transport. These results indicate that the intra-molecular rotation is elastic but proton transport requires more strict subunit/subunit interaction.

A unique F-type H⁺-ATPase from Streptococcus mutans: an active H⁺ pump at acidic pH.

We have shown previously that the Streptococcus mutans F-type H(+)-ATPase (F(O)F(1)) c subunit gene could complement Escherichia coli defective in the corresponding gene, particularly at acidic pH (Araki et al., (2013) [14]). In this study, the entire S. mutans F(O)F(1) was functionally assembled in the E. coli plasma membrane (SF(O)F(1)). Membrane SF(O)F(1) ATPase showed optimum activity at pH 7, essentially the same as that of the S. mutans, although the activity of E. coli F(O)F(1) (EF(O)F(1)) was optimum at pH≥9. The membranes showed detectable ATP-dependent H(+)-translocation at pH 5.5-6.5, but not at neutral conditions (pH≥7), consistent with the role of S. mutans F(O)F(1) to pump H(+) out of the acidic cytoplasm. A hybrid F(O)F(1), consisting of membrane-integrated F(O) and -peripheral F(1) sectors from S. mutans and E. coli (SF(O)EF(1)), respectively, essentially showed the same pH profile as that of EF(O)F(1) ATPase. However, ATP-driven H(+)-transport was similar to that by SF(O)F(1), with activity at acidic pH. Replacement of the conserved c subunit Glu53 in SF(O)F(1) abolished H(+)-transport at pH 6 or 7, suggesting its role in H(+) transport. Mutations in the SF(O)F(1) c subunit, Ser17Ala or Glu20Ile, changed the pH dependency of H(+)-transport, and the F(O) could transport H(+) at pH 7, as the membranes with EF(O)F(1). Ser17, Glu20, and their vicinity were suggested to be involved in H(+)-transport in S. mutans at acidic pH.

Complementation of the Fo c subunit of Escherichia coli with that of Streptococcus mutans and properties of the hybrid FoF1 ATP synthase.

The c subunit of Streptococcus mutans ATP synthase (FoF1) is functionally exchangeable with that of Escherichia coli, since E. coli with a hybrid FoF1 is able to grow on minimum succinate medium through oxidative phosphorylation. E. coli F1 bound to the hybrid Fo with the S. mutans c subunit showed N,N'-dicyclohexylcarbodiimide-sensitive ATPase activity similar to that of E. coli FoF1. Thus, the S. mutans c subunit assembled into a functional Fo together with the E. coli a and b subunits, forming a normal F1 binding site. Although the H(+) pathway should be functional, as was suggested by the growth on minimum succinate medium, ATP-driven H(+) transport could not be detected with inverted membrane vesicles in vitro. This observation is partly explained by the presence of an acidic residue (Glu-20) in the first transmembrane helix of the S. mutans c subunit, since the site-directed mutant carrying Gln-20 partly recovered the ATP-driven H(+) transport. Since S. mutans is recognized to be a primary etiological agent of human dental caries and is one cause of bacterial endocarditis, our system that expresses hybrid Fo with the S. mutans c subunit would be helpful to find antibiotics and chemicals specifically directed to S. mutans.

Halotolerant cyanobacterium Aphanothece halophytica contains an Na+-dependent F1F0-ATP synthase with a potential role in salt-stress tolerance.

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium that can grow in media of up to 3.0 m NaCl and pH 11. Here, we show that in addition to a typical H(+)-ATP synthase, Aphanothece halophytica contains a putative F(1)F(0)-type Na(+)-ATP synthase (ApNa(+)-ATPase) operon (ApNa(+)-atp). The operon consists of nine genes organized in the order of putative subunits ß, ε, I, hypothetical protein, a, c, b, α, and γ. Homologous operons could also be found in some cyanobacteria such as Synechococcus sp. PCC 7002 and Acaryochloris marina MBIC11017. The ApNa(+)-atp operon was isolated from the A. halophytica genome and transferred into an Escherichia coli mutant DK8 (Δatp) deficient in ATP synthase. The inverted membrane vesicles of E. coli DK8 expressing ApNa(+)-ATPase exhibited Na(+)-dependent ATP hydrolysis activity, which was inhibited by monensin and tributyltin chloride, but not by the protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The Na(+) ion protected the inhibition of ApNa(+)-ATPase by N,N'-dicyclohexylcarbodiimide. The ATP synthesis activity was also observed using the Na(+)-loaded inverted membrane vesicles. Expression of the ApNa(+)-atp operon in the heterologous cyanobacterium Synechococcus sp. PCC 7942 showed its localization in the cytoplasmic membrane fractions and increased tolerance to salt stress. These results indicate that A. halophytica has additional Na(+)-dependent F(1)F(0)-ATPase in the cytoplasmic membrane playing a potential role in salt-stress tolerance.

Effects of mutations in the beta subunit hinge domain on ATP synthase F1 sector rotation: interaction between Ser 174 and Ile 163.

A complex of gamma, epsilon, and c subunits rotates in ATP synthase (F(o)F(1)) coupling with proton transport. Replacement of betaSer174 by Phe in beta-sheet4 of the beta subunit (betaS174F) caused slow gamma subunit revolution of the F(1) sector, consistent with the decreased ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the beta subunit, J. Biol. Chem. 282 (2007) 20698-20704]. Modeling of the domain including beta-sheet4 and alpha-helixB predicted that the mutant betaPhe174 residue undergoes strong and weak hydrophobic interactions with betaIle163 and betaIle166, respectively. Supporting this prediction, the replacement of betaIle163 in alpha-helixB by Ala partially suppressed the betaS174F mutation: in the double mutant, the revolution speed and ATPase activity recovered to about half of the levels in the wild-type. Replacement of betaIle166 by Ala lowered the revolution speed and ATPase activity to the same levels as in betaS174F. Consistent with the weak hydrophobic interaction, betaIle166 to Ala mutation did not suppress betaS174F. Importance of the hinge domain [phosphate-binding loop (P-loop)/alpha-helixB/loop/beta-sheet4, betaPhe148-betaGly186] as to driving rotational catalysis is discussed.

Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the beta subunit.

A complex of gamma, epsilon, and c subunits rotates in ATP synthase (FoF(1)) coupled with proton transport. A gold bead connected to the gamma subunit of the Escherichia coli F(1) sector exhibited stochastic rotation, confirming a previous study (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126-4131). A similar approach was taken for mutations in the beta subunit key region; consistent with its bulk phase ATPase activities, F(1) with the Ser-174 to Phe substitution (betaS174F) exhibited a slower single revolution time (time required for 360 degree revolution) and paused almost 10 times longer than the wild type at one of the three 120 degrees positions during the stepped revolution. The pause positions were probably not at the "ATP waiting" dwell but at the "ATP hydrolysis/product release" dwell, since the ATP concentration used for the assay was approximately 30-fold higher than the K(m) value for ATP. A betaGly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of betaS174F. The revertant (betaG149A/betaS174F) exhibited similar rotation to the wild type, except that it showed long pauses less frequently. Essentially the same results were obtained with the Ser-174 to Leu substitution and the corresponding revertant betaG149A/betaS174L. These results indicate that the domain between beta-sheet 4 (betaSer-174) and P-loop (betaGly-149) is important to drive rotation.

ATP-dependent rotation of mutant ATP synthases defective in proton transport.

During ATP hydrolysis, the gammaepsilon c10 complex (gamma and epsilon subunits and a c subunit ring formed from 10 monomers) of F0F1 ATPase (ATP synthase) rotates relative to the alpha3beta3delta ab2 complex, leading to proton transport through the interface between the a subunit and the c subunit ring. In this study, we replaced the two pertinent residues for proton transport, cAsp-61 and aArg-210 of the c and a subunits, respectively. The mutant enzymes exhibited lower ATPase activities than that of the wild type but exhibited ATP-dependent rotation in planar membranes, in which their original assemblies are maintained. The mutant enzymes were defective in proton transport, as shown previously. These results suggest that proton transport can be separated from rotation in ATP hydrolysis, although rotation ensures continuous proton transport by bringing the cAsp-61 and aArg-210 residues into the correct interacting positions.

Subunit rotation of vacuolar-type proton pumping ATPase: relative rotation of the G and C subunits.

Vacuolar-type ATPases V1V0 (V-ATPases) are found ubiquitously in the endomembrane organelles of eukaryotic cells. In this study, we genetically introduced a His tag and a biotin tag onto the c and G subunits, respectively, of Saccharomyces cerevisiae V-ATPase. Using this engineered enzyme, we observed directly the continuous counter-clockwise rotation of an actin filament attached to the G subunit when the enzyme was immobilized on a glass surface through the c subunit. V-ATPase generated essentially the same torque as the F-ATPase (ATP synthase). The rotation was inhibited by concanamycin and nitrate but not by azide. These results demonstrated that the V- and F-ATPase carry out a common rotational catalysis.

Subunit rotation of ATP synthase embedded in membranes: a or beta subunit rotation relative to the c subunit ring.

ATP synthase F(o)F(1) (alpha(3)beta(3)gammadelta epsilon ab(2)c(10-14)) couples an electrochemical proton gradient and a chemical reaction through the rotation of its subunit assembly. In this study, we engineered F(o)F(1) to examine the rotation of the catalytic F(1) beta or membrane sector F(o) a subunit when the F(o) c subunit ring was immobilized; a biotin-tag was introduced onto the beta or a subunit, and a His-tag onto the c subunit ring. Membrane fragments were obtained from Escherichia coli cells carrying the recombinant plasmid for the engineered F(o)F(1) and were immobilized on a glass surface. An actin filament connected to the beta or a subunit rotated counterclockwise on the addition of ATP, and generated essentially the same torque as one connected to the c ring of F(o)F(1) immobilized through a His-tag linked to the alpha or beta subunit. These results established that the gamma epsilon c(10-14) and alpha(3)beta(3)deltaab(2) complexes are mechanical units of the membrane-embedded enzyme involved in rotational catalysis.