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• W2005059216 abstract "The ϵ subunit of F1-ATPase from the thermophilic Bacillus PS3 (TF1) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the ϵ subunit remains to be determined. To address this question, 11 mutants of the ϵ subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the α3β3γ complex of TF1. The ATPase activity of the resulting α3β3γϵ complexes was measured, and the extent of inhibition by the mutant ϵ subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the ϵ subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF1 by fixing the ϵ subunit into the folded conformation. The ϵ subunit of F1-ATPase from the thermophilic Bacillus PS3 (TF1) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the ϵ subunit remains to be determined. To address this question, 11 mutants of the ϵ subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the α3β3γ complex of TF1. The ATPase activity of the resulting α3β3γϵ complexes was measured, and the extent of inhibition by the mutant ϵ subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the ϵ subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF1 by fixing the ϵ subunit into the folded conformation. F0F1-ATPase/synthase (F0F1) catalyzes ATP synthesis via coupling of the proton flow driven by the electrochemical gradient of protons or sodium. F0F1 consists of two rotary molecular motors: a water-soluble, ATP-driven F1 motor and a membrane-embedded, H+- or Na+-driven F0 motor. These molecular motors are connected together to couple ATP synthesis/hydrolysis and ion flow (1Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1595) Google Scholar, 2Kinosita Jr., K. Yasuda R. Noji H. Essays Biochem. 2000; 35: 3-18Crossref PubMed Scopus (40) Google Scholar, 3Yoshida M. Muneyuki E. Hisabori T. Nat. Rev. Mol. Cell Biol. 2001; 2: 669-677Crossref PubMed Scopus (713) Google Scholar, 4Senior A.E. Nadanaciva S. Weber J. Biochim. Biophys. Acta. 2002; 1553: 188-211Crossref PubMed Scopus (340) Google Scholar). The F1-ATPase (α3β3δγϵ) hydrolyzes ATP into ADP and inorganic phosphate, and the hydrolysis of one ATP drives the discrete 120° rotation of the γϵ subunits relative to the other subunits (5Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1966) Google Scholar, 6Yasuda R. Noji H. Kinosita Jr., K. Yoshida M. Cell. 1998; 93: 1117-1124Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). As the smallest subunit of F1-ATPase, the ϵ subunit acts as an endogenous inhibitor of the ATPase activity in both the bacterial and chloroplast F1-ATPase, where it is believed to play a regulatory role in ATP synthase (7Smith J.B. Sternweis P.C. Heppel L.A. J. Supramol. Struct. 1975; 3: 248-255Crossref PubMed Scopus (48) Google Scholar, 8Laget P.P. Smith J.B. Arch. Biochem. Biophys. 1979; 197: 83-89Crossref PubMed Scopus (70) Google Scholar, 9Ort D.R. Oxborough K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1992; 43: 269-291Crossref Scopus (80) Google Scholar, 10Kato Y. Matsui T. Tanaka N. Muneyuki E. Hisabori T. Yoshida M. J. Biol. Chem. 1997; 272: 24906-24912Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). A recent single molecule study revealed its importance in efficient coupling in rotation and ATP synthesis (11Rondelez Y. Tresset G. Nakashima T. Kato-Yamada Y. Fujita H. Takeuchi S. Noji H. Nature. 2005; 433: 773-777Crossref PubMed Scopus (303) Google Scholar). The ϵ subunit consists of two distinct domains, an N-terminal β sandwich domain and a C-terminal α helical domain. Structural and biochemical studies have shown that the ϵ subunit adopts at least two different conformations in F1 and F0F1 (10Kato Y. Matsui T. Tanaka N. Muneyuki E. Hisabori T. Yoshida M. J. Biol. Chem. 1997; 272: 24906-24912Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 12Gibbons C. Montgomery M.G. Leslie A.G.W. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (436) Google Scholar, 13Rodgers A.J.W. Wilce M.C.J. Nat. Struct. Biol. 2000; 7: 1051-1054Crossref PubMed Scopus (153) Google Scholar, 14Richter M.L. McCarty R.E. J. Biol. Chem. 1987; 262: 15037-15040Abstract Full Text PDF PubMed Google Scholar, 15Kato-Yamada Y. Bald D. Koike M. Motohashi K. Hisabori T. Yoshida M. J. Biol. Chem. 1999; 274: 33991-33994Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 17Tsunoda S.P. Rodgers A.J.W. Aggeler R. Wilce M.C.J. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6560-6564Crossref PubMed Scopus (163) Google Scholar, 18Suzuki T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 19Iino R. Murakami T. Iizuka S. Kato-Yamada Y. Suzuki T. Yoshida M. J. Biol. Chem. 2005; 280: 40130-40134Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The conformation that results in the inhibition of ATPase exists in an extended state, in which the C-terminal helical domain of the ϵ subunit unfolds to run parallel to the γ subunit. The conformation in which ATPase is active is known as the folded state and is characterized by C-terminal α helices folded into a hairpin configuration. The conformational change of the ϵ subunit is controlled by the concentration of both ATP and ADP as well as the membrane potential (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 17Tsunoda S.P. Rodgers A.J.W. Aggeler R. Wilce M.C.J. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6560-6564Crossref PubMed Scopus (163) Google Scholar, 18Suzuki T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 19Iino R. Murakami T. Iizuka S. Kato-Yamada Y. Suzuki T. Yoshida M. J. Biol. Chem. 2005; 280: 40130-40134Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The isolated ϵ subunit of F1 from the thermophilic Bacillus strain PS3 (TF1) 2The abbreviations used are: TF1F1-ATPase from thermophilic Bacillus PS3BF1F1-ATPase from B. subtilis;EF1, F1-ATPase from E. coliIC3-PE-maleimideN-ethyl-N′-{5-[N″-(2-maleimidoethyl)pyperazinocarbonyl]pentyl} indocarbocyanineWTwild typeTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidAMP-PNP5′-adenylyl-β,γ-imidodiphosphate was recently found to bind ATP (20Kato-Yamada Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Binding was so specific that GTP and ADP failed to form a complex with the ϵ subunit, as assayed by gel filtration. These results led to the suggestion that the ϵ subunit is both a regulator and sensor of cellular ATP concentration. ATP binding was also observed with the ϵ subunits of F1-ATPases from Bacillus subtilis (21Kato-Yamada Y. FEBS Lett. 2005; 579: 6875-6878Crossref PubMed Scopus (40) Google Scholar) and Escherichia coli (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). X-ray crystallographic analysis revealed that ATP is bound to the ϵ subunit in the folded state (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). As the ATP binding site consists of residues from the N-terminal domain and the first and second α helices of the C-terminal domain, ATP may bind to the ϵ subunit alone in the folded state. An NMR study revealed that in the absence of ATP, the ϵ subunit adopts a different conformation. ATP may stabilize the folded conformation of the ϵ subunit (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). ATP binding was also observed with the ϵ subunit in the γϵ subcomplex, indicating that ATP binding to the ϵ subunit can occur in ATP synthase, in which it may play a regulatory role (23Iizuka S. Kato S. Yoshida M. Kato-Yamada Y. Biochem. Biophys. Res. Commun. 2006; 349: 1368-1371Crossref PubMed Scopus (11) Google Scholar). F1-ATPase from thermophilic Bacillus PS3 F1-ATPase from B. subtilis;EF1, F1-ATPase from E. coli N-ethyl-N′-{5-[N″-(2-maleimidoethyl)pyperazinocarbonyl]pentyl} indocarbocyanine wild type 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid 5′-adenylyl-β,γ-imidodiphosphate To determine the role of ATP binding to the ϵ subunit in the regulation of the F0F1-ATP synthase, 11 mutants of the ϵ subunit were constructed, in which basic or acidic residues were substituted with alanine residues. Several mutants with impaired ATP binding were further reconstituted with α3β3γ, and the ATPase activities of these α3β3γϵ complexes were measured. The results clearly show that ATP binding to the ϵ subunit affects the regulation of ATPase activity. Materials—Wild-type and αK175A/T176A mutant (noncatalytic site-deficient mutant, ΔNC) 3As noted in Ref. 26Ono S. Hara K.Y. Hirao J. Matsui T. Noji H. Yoshida M. Muneyuki E. Biochim. Biophys. Acta. 2003; 1607: 35-44Crossref PubMed Scopus (27) Google Scholar, αK175A/T176A mutations are enough to suppress the nucleotide binding to the noncatalytic site. α3β3γ complexes of TF1 were prepared as described previously (24Matsui T. Yoshida M. Biochim. Biophys. Acta. 1995; 1231: 139-146Crossref PubMed Scopus (86) Google Scholar, 25Matsui T. Muneyuki E. Honda M. Allison W.S. Dou C. Yoshida M. J. Biol. Chem. 1997; 272: 8215-8221Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). An expression plasmid for a mutant α3β3γ complex was prepared containing KT/AA substitutions in Walker A motifs (27Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4257) Google Scholar) in both the α and β subunits (αK175A/T176A and βK164A/T165A, noncatalytic and catalytic site-deficient mutants, ΔNC/ΔC); a DNA fragment containing the βK164A/T165A mutations was prepared using the overlap extension PCR method (28Higuchi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2102) Google Scholar, 29Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar) applied to the expression plasmid for the wild-type (WT) α3β3γ complex. A 900-bp fragment containing mutations was treated with restriction enzymes AgeI and MunI, and the resulting 624-bp fragment was purified and directly transferred to the respective sites of an expression plasmid for the ΔNC (αK175A/T176A) mutant α3β3γ complex. The mutant α3β3γ complex was purified in the same way as the wild-type complex. The wild-type and S48C/N125C mutant (ϵNCX, a mutant ϵ subunit of TF1 in which N- and C-terminal domains can be cross-linked) ϵ subunits were prepared as previously described (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 30Hisabori T. Kato Y. Motohashi K. Kroth-Pancic P. Strotmann H. Amano T. Eur. J. Biochem. 1997; 247: 1158-1165Crossref PubMed Scopus (35) Google Scholar). The expression plasmids for the mutant ϵ subunits were prepared by the overlap extension PCR method or mega-primer PCR method (31Landt O. Grunert H.P. Hahn U. Gene (Amst.). 1990; 96: 125-128Crossref PubMed Scopus (639) Google Scholar) applied to the pET21/TF1-ϵ (wild-type) plasmid (20Kato-Yamada Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The Q107C mutation for fluorescent labeling was introduced by the mega-primer PCR method applied to the expression plasmid for the mutants deficient in ATP binding. Mutations were confirmed by DNA sequencing. Except for the E83A and E83A/Q107C mutants, the ϵ subunits were prepared as described previously (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 30Hisabori T. Kato Y. Motohashi K. Kroth-Pancic P. Strotmann H. Amano T. Eur. J. Biochem. 1997; 247: 1158-1165Crossref PubMed Scopus (35) Google Scholar). It was noticed that the use of a large sized butyl-TOYOPEARL column resulted in a reduced amount of bound ATP in the ϵ subunit preparation, reaching <0.05 mol/mol (23Iizuka S. Kato S. Yoshida M. Kato-Yamada Y. Biochem. Biophys. Res. Commun. 2006; 349: 1368-1371Crossref PubMed Scopus (11) Google Scholar). The E83A mutant ϵ subunit was prepared as follows. Cells were suspended in 50 mm Tris-HCl (pH 8) and 1 mm EDTA (buffer A) and disrupted by French press. Cell lysate was ultracentrifuged at 220,000 × g for 1 h at 4 °C. The supernatant was applied to a DEAE-TOYOPEARL column equilibrated with the same buffer. As the mutant ϵ subunit was adsorbed on the column, a 0–100 mm linear gradient of NaCl was applied, and the fractions containing ϵ subunit were pooled. Solid ammonium sulfate was added to the sample to 20% saturation, and the sample was applied to a phenyl-TOYOPEARL column equilibrated with buffer A containing a 25% saturated concentration of ammonium sulfate. Proteins were eluted with a 25 to 0% saturation linear gradient of ammonium sulfate. For purification of the E83A/Q107C mutant, 1 mm dithiothreitol was included in all buffers. The α3β3γϵ complex was prepared by mixing the purified α3β3γ complex and ϵ subunit to a molar ratio of 1:10 (10Kato Y. Matsui T. Tanaka N. Muneyuki E. Hisabori T. Yoshida M. J. Biol. Chem. 1997; 272: 24906-24912Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Excess ϵ subunit was not removed for the ATPase measurements. For the experiments in Fig. 6, excess ϵ subunit was removed from the α3β3γϵ complex by three successive ultrafiltrations (100-kDa cutoff) with an Amicon centrifugal filter device as described previously (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Formation of the α3β3γϵ complex was confirmed by polyacrylamide gel electrophoresis without a denaturing reagent. ATP Binding Assay—ATP binding assay by gel-filtration chromatography on a Sephadex G25F column (GE Healthcare) was performed as reported previously in a buffer consisting of 50 mm Tris-HCl (pH 8) and 100 mm KCl at room temperature (25 °C) (20Kato-Yamada Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). ATP binding assay by fluorescence was performed as described above (19Iino R. Murakami T. Iizuka S. Kato-Yamada Y. Suzuki T. Yoshida M. J. Biol. Chem. 2005; 280: 40130-40134Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) except that N-ethyl-N′-{5-[N″-(2-maleimidoethyl)piperazinocarbonyl]pentyl}indocarbocyanine (IC3-PE-maleimide; Dojin) was used instead of Cy3-maleimide. Labeling was performed in 50 mm TES-NaOH (pH 7) and 100 mm NaCl with a protein-to-dye ratio of 1:2–7. Excess dye was removed with a Bio-Gel P6 centrifuge column (Bio-Rad). The assay mixture contained 50 mm HEPES-KOH (pH 7.5), 100 mm KCl, and 10 mm MgCl2. Fluorescently labeled ϵ subunit (100 nm) was incubated in the assay mixture, and the changes in fluorescence were measured upon addition of ATP. The measurements were performed at 25 °C with an FP-6500 fluorescent spectrometer (JASCO). Excitation and emission wavelengths were 522 and 559 nm, respectively. Slit widths for excitation and emission were 3 nm. Fluorescent change was corrected for corresponding measurement without ATP and plotted against ATP concentration. ATPase Assay—ATPase activity was measured spectrophotometrically with an NADH-coupled ATP-regenerating system at 25 °C (32Stiggal D.L. Galante Y.M. Hatefi Y. Methods Enzymol. 1979; 55: 308-315Crossref PubMed Scopus (78) Google Scholar). The assay mixture consisted of 50 mm Tris-HCl (pH 8), 100 mm KCl, 2.5 mm phosphoenolpyruvate, 2 mm MgCl2, 0.2 mm NADH, 50 μg/ml pyruvate kinase, 50 μg/ml lactate dehydrogenase, and the indicated concentration of ATP-Mg. The reaction was initiated by the addition of 10 nm TF1 α3β3γϵ complex to the assay mixture, and changes in absorbance at 340 nm were measured in a V-550 or V-530 spectrophotometer (JASCO). Velocity measurements were taken at 540–1140 s after the start of the reactions. One unit of ATPase activity was defined as that producing 1 μmol of ADP/min. Detection of Conformation of the ϵ Subunit—The detection of a conformational change in the ϵ subunit corresponding to the formation of an intramolecular disulfide bond was performed in essentially the same way as described previously (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Reduced ΔNC/ΔC-α3β3γϵNCX and ΔNC-α3β3γϵNCX complexes were incubated with or without 2 mm ATP-Mg. After 10 min, 0, 10, 20, or 50 μm CuCl2 was added, and the sample was incubated for 1 h at room temperature. The cross-linking reaction was quenched by addition of 10 mm EDTA. IC3-PE-maleimide (500 μm) and 0.1% SDS were then added to the mixture and incubated for 10 min at room temperature to label cysteine residues that had not formed a disulfide bond. The reaction was quenched by the addition of 12 mm N-ethylmaleimide. The samples were analyzed by SDS-PAGE without reducing reagent (15% polyacrylamide). The fluorescent image was taken by a Typhoon 9210 image analyzer (GE Healthcare) with a green laser (532 nm) and an appropriate filter set. The gel was then stained with Coomassie Brilliant Blue R-250, and the image was recorded with a GT-9800F flatbed scanner (Epson). As judged by the gel-filtration analysis, no difference in ATP binding was observed between the mutant ϵNCX subunit and the wild-type (data not shown). Re-inactivation Assay—Re-inactivation of the α3β3γϵ complex by lowering the ATP concentration was measured as follows. The α3β3γϵ complex was incubated with 4 mm ATP-Mg in a buffer consisting of 50 mm Tris-HCl (pH 8), 100 mm KCl, and 2 mm MgCl2 at 25 °C for more than 10 min. Bovine serum albumin (0.1 mg/ml) was included in the control measurement, which was carried out with the α3β3γ complex. Five μl of the mixture was then added to 1 ml of ATPase assay mixture without ATP in a quartz cuvette to give a final concentration of ATP at 20 μm. Other Methods—Protein concentrations of the ϵ subunits were determined by the method of Bradford (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar) using bovine serum albumin as a standard and corrected by multiplying by a factor of 0.54 according to the results of amino acid quantification (20Kato-Yamada Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Concentrations of α3β3γ or α3β3γϵ complexes were determined by measuring UV absorption spectra using A280 = 0.45 at 1 mg/ml (25Matsui T. Muneyuki E. Honda M. Allison W.S. Dou C. Yoshida M. J. Biol. Chem. 1997; 272: 8215-8221Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Chemicals were of the highest grades available. Altered ATP Binding to the Mutant ϵ Subunits—To obtain ϵ subunit mutants that showed altered ATP binding, alanine-based substitutions were carried out (highlighted in Fig. 1A) for the following reasons: 1) These are the basic or acidic residues present in the C-terminal domain that are different in TF1 and B. subtilis F1-ATPase (BF1). Although ATP binding to the ϵ subunit of BF1 was observed, the affinity for ATP was greatly reduced (21Kato-Yamada Y. FEBS Lett. 2005; 579: 6875-6878Crossref PubMed Scopus (40) Google Scholar). One possibility is that these residues may be responsible for the difference (R84A, K94A, E98A, and R102A, shown in blue in Fig. 1B). 2) These are the residues that formed hydrogen bonds with ATP in the crystal structure of the TF1 ϵ subunit-ATP complex (E83A, D89A, R92A, R99A, R115A, R122A, and R126A, marked with asterisks in Fig. 1A and shown in red in Fig. 1B), although two of these (Arg122 and Arg126) were thought to play an inhibitory role in a previous study (34Hara K.Y. Kato-Yamada Y. Kikuchi Y. Hisabori T. Yoshida M. J. Biol. Chem. 2001; 276: 23969-23973Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Arg115 appeared to interact with ATP bound to another monomer ϵ subunit in the crystal structure (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar) and, possibly, to be involved in ATP binding. ATP binding to these mutants was assayed by gel-filtration chromatography. The results are shown in Fig. 2. Two mutants (R102A and R115A) showed essentially the same profile as the wild type. Four (E83A, R92A, R122A, and R126A) showed no ATP binding, and the other five (R84A, D89A, K94A, E98A, and R99A) showed a moderate degree of binding. For further analyses, four mutants (E83A, R92A, R122A, and R126A) were chosen that showed virtually no ATP binding in the gel-filtration assay. Interestingly, all these residues are conserved between TF1 and BF1. Arg92, which was included in the previously proposed consensus ATP binding motif, I(L)DXXRA, was found to be important for ATP binding (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). As determined previously, Glu83 was also found to be important for ATP binding (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). The mutants selected by the differences between TF1 and BF1 had a relatively minor effect on ATP binding. The difference in ATP binding between TF1 and BF1 may be because of the differences in other residues. As for E. coli F1-ATPase (EF1) ϵ, only Arg92 is conserved, although EF1 ϵ possesses a lysine residue in place of Arg122. These differences may be the cause of the very weak affinity for ATP of the EF1 ϵ subunit (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). The cysteine residue introduced at Gln107, located in the loop region between the two C-terminal α helices, was labeled with fluorescent maleimide. This was used to provide a more accurate measurement of the ATP binding affinity of the mutants that showed no ATP binding in the gel-filtration analysis. Although the extent of fluorescence varied with mutants, possibly because of the differences in the interactions between dye and protein, saturating ATP concentration dependence of the fluorescence was observed (Fig. 3). The Kd for ATP at 25 °C of these mutants varied from 100 μm to >1 mm. Although the crystal structure suggests that the Glu83 and Arg92 residues are important for ATP binding, the mutation at Arg126 was shown to have a more significant effect.FIGURE 2Gel-filtration analysis of ATP binding to the mutant ϵ subunits of TF1. ATP binding to the mutant ϵ subunits (25 μm) was analyzed on a Sephadex G25F column. Elution was monitored at 260 nm. Mutations are described on the left of each trace. Upper and lower traces represent measurements with and without 25 μm ATP, respectively. Positions of the peaks containing ϵ subunit are marked with dotted lines. The vertical bar on top of the figure represents 0.01 absorbance unit at 260 nm. Other experimental conditions are described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Affinity of the mutant ϵ subunits for ATP. Changes in the fluorescence of IC3-labeled mutant ϵ subunits upon addition of ATP were measured. ATP was added to the IC3-labeled ϵ subunit sequentially. Changes in fluorescence are corrected for corresponding measurements without ATP, normalized to initial fluorescence, and plotted against ATP concentration. Excitation and emission wavelengths were 522 and 559 nm, respectively. Open square, open circle, closed circle, open diamond, and closed diamond represent wild type, E83A, R92A, R122A, and R126A, respectively. Lines represent fits with data by a simple binding scheme. Calculated Kd values and maximum fluorescence of the mutants for ATP are as follows: 4.3 μm, 79% (wild type); 520 μm, 50% (E83A); 160 μm, 63% (R92A); 280 μm, 46% (R122A); and >1000 μm, 61% (R126A), respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Suppressed ATPase Activity with Mutant ϵ Subunits—There was no significant difference in ATPase activities of α3β3γϵ complexes containing mutant ϵ subunit at high ATP concentration (2 mm) (Fig. 4A). However, reduction in ATP concentration resulted in more prominent differences in mutant ATPase activities. For example, at 200 μm ATP (Fig. 4B), α3β3γϵ complexes containing R126A showed markedly lower ATPase activity than the wild-type complex. At 20 μm ATP (Fig. 4C), the differences were more significant, and the α3β3γϵ complex containing E83A or R92A also showed a reduction in ATPase activity compared with the wild-type complex. The ATP concentration dependence of the ATPase activity is summarized in Fig. 5. The ATP concentration that resulted in half-maximum activity may not directly relate to the Kd for ATP of the ϵ subunit because activity was measured at a defined time point and not at final equilibrium. However, with the exception of R122A, increased inhibition was observed with less ATP binding mutant ϵ subunit (Fig. 5B). The strengths of ATP binding were in the following order: WT > R92A > R122A > E83A > R126A, whereas the activities were in the following order: R122A > WT > R92A > E83A > R126A. The exceptional behavior of R122A may be because of the direct involvement of Arg122 in the inhibitory effect of the ϵ subunit itself (34Hara K.Y. Kato-Yamada Y. Kikuchi Y. Hisabori T. Yoshida M. J. Biol. Chem. 2001; 276: 23969-23973Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar).FIGURE 5ATP concentration dependence of the ATPase activity. A, ATPase activities of the α3β3γϵ complexes are plotted against ATP concentration. Velocities were taken at 540–1140 s after the initiation of the reactions. Closed square, open square, open circle, closed circle, open diamond, and closed diamond represent α3β3γϵ complexes with mutant ϵ subunits: none (α3β3γ complex), wild type, E83A, R92A, R122A, and R126A, respectively. B, ATPase activities of the α3β3γϵ complexes are normalized to that of the α3β3γ complex at each ATP concentration. Symbols are the same as in A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ATP Binding to the ϵ Subunit Is Not a Primary Event in Activation—In a previous study, nucleotide binding to the β subunit was found to be responsible for the conformational change in the ϵ subunit because the conformational change was also observed in the noncatalytic site-deficient mutant (ΔNC); in addition, it was noted that AMP-PNP could also induce conformational change in the ϵ subunit (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). However, the finding that the ϵ subunit can bind ATP raises the possibility that ATP binding may be a trigger for the conformational change in the ϵ subunit. To test this hypothesis, a mutant α3β3γ complex was prepared that contained KT/AA substitutions in Walker A motifs (27Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4257) Google Scholar) in both the α and β subunits (ΔNC/ΔC). The KT/AA mutations are known to result in the loss of ATP binding in several Walker-type ATPases (25Matsui T. Muneyuki E. Honda M. Allison W.S. Dou C. Yoshida M. J. Biol. Chem. 1997; 272: 8215-8221Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 35Watanabe Y.-H. Motohashi K. Yoshida M. J. Biol. Chem. 2002; 277: 5804-5809Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The ΔNC/ΔC-α3β3γ complex showed no steady-state ATPase activity (that is, less than 1/100 of ΔNC, i.e. <1/10000 of the wild type) despite the presence of 0.3% lauryldimethylamine oxide. This suggests that virtually no ATP was bound to the catalytic sites (data not shown). In Fig. 6, a decrease in the fluorescent band indicated that the ϵ subunit was adopting a folded state, thus easily forming a cross-link between S48C and N125C. With the ΔNC mutant, ATP-dependent enhancement of the cross-link formation in the ϵNCX subunit was observed (Fig. 6) (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In contrast, no enhancement of cross-link formation was observed with the ΔNC/ΔC mutant (Fig. 6). The results implied that ATP does not bind primarily to the ϵ subunit, but rather to the (possibly second or third) catalytic site(s). This may be the trigger for activation from inhibition by the ϵ subunit. When the ϵ subunit is in the extended state, the ATP binding site is divided, and ATP cannot access the residues therein as they are hidden within the α3β3 cavity. ATP binding to the β subunit may induce conformational changes in the β subunit, resulting in expulsion of the ϵ subunit from the α3β3 cylinder. Once the C-terminal helices are expelled from the α3β3 cylinder, ATP can access the binding site on the ϵ subunit. Re-inactivation by Decreasing ATP Concentration—At high concentrations of ATP (e.g. 2 mm) (Fig. 4A), the ATPase activities of the α3β3γϵ complexes containing the wild-type or mutant ϵ subunit were found to be similar. However, the ATPase activities differed significantly at low concentrations of ATP (e.g. 20 μm) (Fig. 4C). Following from this, the ATP concentration was changed from high to low to determine the impact of ATP dissociation from the ϵ subunit in the α3β3γϵ complex. The α3β3γϵ complex was incubated with 4 mm ATP-Mg for more than 10 min at room temperature. As reported previously, this treatment activated the α3β3γϵ complex (10Kato Y. Matsui T. Tanaka N. Muneyuki E. Hisabori T. Yoshida M. J. Biol. Chem. 1997; 272: 24906-24912Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Five μl of the sample was then injected into 1 ml of ATP-free ATPase assay mixture to give a final ATP concentration of 20 μm. In contrast to the ATPase measurement taken without preincubation with ATP (Fig. 4C), the WT α3β3γϵ complex showed similar ATPase activity to the α3β3γ complex (Fig. 7). Slow inactivation was observed with E83A, R92A, and R126A, for which the Kd for ATP was higher than 20 μm. The reason ATPase activity remained high in the WT α3β3γϵ complex may have been because the wild-type ϵ subunit maintained high affinity for ATP, and thus ATP remained bound even at a concentration of 20 μm. ATP Binding to the ϵ Subunit Shifts the Equilibrium between Inactive and Active States—The ATP binding form of the ϵ subunit was revealed as the folded state (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). The binding site consists of both the N- and C-terminal domains. Moreover, there may be no room for ATP to bind to the ϵ subunit in the extended form, as the C-terminal helices of the ϵ subunit are surrounded by the α, β, and γ subunits. The results also imply that fluctuation of the ϵ subunit between folded and extended forms rarely occurs in the absence of ATP binding to the β subunits. The likely role of ATP binding to the ϵ subunit is to stabilize the ϵ subunit of activated complex in the folded state. A simplified model is shown in Fig. 8. The ATPase-inactive TF1 complex is activated by binding of ATP to the β subunit(s). Upon activation, the ϵ subunit changes its conformation from the extended to the folded state. However, in the absence of bound ATP, the folded state is unstable (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar), and the C-terminal domain of the ϵ subunit has the tendency to return to its extended form. ATP binding to the ϵ subunit stabilizes the folded state, shifts the equilibrium between the inactive and active states of TF1, and results in a higher proportion of active complex. This may be important when, for example, cells contain sufficient ATP, but the membrane potential is low because of an alkaline environment. Under such conditions, F0F1 may work as a proton pump to build up membrane potential at the expense of ATP. In the absence of ATP binding to the ϵ subunit, however, F0F1 may readily adopt the ATPase-inactive conformation even if the cell contains sufficient ATP. It should be noted that affinity for ATP of the ϵ subunit under physiological temperature is relatively low (Kd = 0.6 mm at 65 °C), and the cellular ATP concentration may fluctuate in this range (19Iino R. Murakami T. Iizuka S. Kato-Yamada Y. Suzuki T. Yoshida M. J. Biol. Chem. 2005; 280: 40130-40134Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In the case of BF1, because the Kd for ATP is in the order of mm (21Kato-Yamada Y. FEBS Lett. 2005; 579: 6875-6878Crossref PubMed Scopus (40) Google Scholar), the regulatory mechanism may be the same as that of TF1. As for EF1, the Kd value of ϵ was reported to be 22 mm at 25 °C (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar), and this low affinity for ATP may be the cause of the strong inhibitory effect of EF1 ϵ compared with that of TF1 ϵ. In summary, the current study shows that the ϵ subunit works not only as a regulator but also as an ATP sensor to regulate enzyme activity according to cellular energy status. Studies on the role of ATP binding to the ϵ subunit in the ATP synthesis reaction are currently under way. We thank Drs. Hiromasa Yagi, Hideaki Tanaka, Tomitake Tsukihara, and Hideo Akutsu and Nobumoto Kajiwara for providing structural information on the ATP binding site of the TF1 ϵ subunit." @default.
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• W2005059216 title "Role of the ϵ Subunit of Thermophilic F1-ATPase as a Sensor for ATP" @default.
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