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W1963539648 abstract "The C-terminal two α-helices of the ϵ-subunit of thermophilic Bacillus FoF1-ATP synthase (TFoF1) adopt two conformations: an extended long arm (“up-state”) and a retracted hairpin (“down-state”). As ATP becomes poor, ϵ changes the conformation from the down-state to the up-state and suppresses further ATP hydrolysis. Using TFoF1 expressed in Escherichia coli, we compared TFoF1 with up- and down-state ϵ in the NTP (ATP, GTP, UTP, and CTP) synthesis reactions. TFoF1 with the up-state ϵ was achieved by inclusion of hexokinase in the assay and TFoF1 with the down-state ϵ was represented by ϵΔc-TFoF1, in which ϵ lacks C-terminal helices and hence cannot adopt the up-state under any conditions. The results indicate that TFoF1 with the down-state ϵ synthesizes GTP at the same rate of ATP, whereas TFoF1 with the up-state ϵ synthesizes GTP at a half-rate. Though rates are slow, TFoF1 with the down-state ϵ even catalyzes UTP and CTP synthesis. Authentic TFoF1 from Bacillus cells also synthesizes ATP and GTP at the same rate in the presence of adenosine 5′-(β,γ-imino)triphosphate (AMP-PNP), an ATP analogue that has been known to stabilize the down-state. NTP hydrolysis and NTP-driven proton pumping activity of ϵΔc-TFoF1 suggests similar modulation of nucleotide specificity in NTP hydrolysis. Thus, depending on its conformation, ϵ-subunit modulates substrate specificity of TFoF1. The C-terminal two α-helices of the ϵ-subunit of thermophilic Bacillus FoF1-ATP synthase (TFoF1) adopt two conformations: an extended long arm (“up-state”) and a retracted hairpin (“down-state”). As ATP becomes poor, ϵ changes the conformation from the down-state to the up-state and suppresses further ATP hydrolysis. Using TFoF1 expressed in Escherichia coli, we compared TFoF1 with up- and down-state ϵ in the NTP (ATP, GTP, UTP, and CTP) synthesis reactions. TFoF1 with the up-state ϵ was achieved by inclusion of hexokinase in the assay and TFoF1 with the down-state ϵ was represented by ϵΔc-TFoF1, in which ϵ lacks C-terminal helices and hence cannot adopt the up-state under any conditions. The results indicate that TFoF1 with the down-state ϵ synthesizes GTP at the same rate of ATP, whereas TFoF1 with the up-state ϵ synthesizes GTP at a half-rate. Though rates are slow, TFoF1 with the down-state ϵ even catalyzes UTP and CTP synthesis. Authentic TFoF1 from Bacillus cells also synthesizes ATP and GTP at the same rate in the presence of adenosine 5′-(β,γ-imino)triphosphate (AMP-PNP), an ATP analogue that has been known to stabilize the down-state. NTP hydrolysis and NTP-driven proton pumping activity of ϵΔc-TFoF1 suggests similar modulation of nucleotide specificity in NTP hydrolysis. Thus, depending on its conformation, ϵ-subunit modulates substrate specificity of TFoF1. IntroductionFoF1-ATP synthase (FoF1) 2The abbreviations used are: FoF1, FoF1-ATP synthase; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; CDTA, 1,2-cyclohexanediaminetetra-acetic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; TFoF1, FoF1-ATP synthase from thermophilic Bacillus PS3; NTP, four kinds nucleotide triphosphates (ATP, GTP, UTP, CTP); NDP, four kinds nucleotide diphosphates (ADP, GDP, UDP, CDP); IMV, inverted membrane vesicle. is ubiquitously found in membranes of bacteria, chloroplast, and mitochondria and synthesizes ATP by the energy of proton flow driven by the proton motive force. FoF1 also is able to catalyze the reverse reaction, ATP hydrolysis-driven proton pumping, which actually occurs in some cases and conditions. FoF1 is a motor enzyme composed of two rotary motors, membrane integral Fo, which converts the proton motive force into rotation, and water-soluble F1, which converts the rotation into synthesis of ATP (1Yoshida M. Muneyuki E. Hisabori T. Nat. Rev. Mol. Cell Biol. 2001; 2: 669-677Crossref PubMed Scopus (699) Google Scholar, 2Boyer P.D. J. Biol. Chem. 2002; 277: 39045-39061Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 3Senior A.E. Nadanaciva S. Weber J. Biochim. Biophys. Acta. 2002; 1553: 188-211Crossref PubMed Scopus (331) Google Scholar, 4Kinosita Jr., K. Adachi K. Itoh H. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 245-268Crossref PubMed Scopus (161) Google Scholar, 5Nakamoto R.K. Baylis Scanlon J.A. Al-Shawi M.K. Arch. Biochem. Biophys. 2008; 476: 43-50Crossref PubMed Scopus (127) Google Scholar, 6Hong S. Pedersen P.L. Microbiol. Mol. Biol. Rev. 2008; 72: 590-641Crossref PubMed Scopus (217) Google Scholar, 7Junge W. Sielaff H. Engelbrecht S. Nature. 2009; 459: 364-370Crossref PubMed Scopus (289) Google Scholar, 8Düser M.G. Zarrabi N. Cipriano D.J. Ernst S. Glick G.D. Dunn S.D. Börsch M. EMBO J. 2009; 28: 2689-2696Crossref PubMed Scopus (95) Google Scholar, 9von Ballmoos C. Wiedenmann A. Dimroth P. Annu. Rev. Biochem. 2009; 78: 649-672Crossref PubMed Scopus (261) Google Scholar, 10Nakanishi-Matsui M. Sekiya M. Nakamoto R.K. Futai M. Biochim. Biophys. Acta. 2010; 1797: 1343-1352Crossref PubMed Scopus (74) Google Scholar). F1 has a subunit composition of α3β3γδϵ and acts as ATPase when isolated. It has been known for the typical bacterial enzymes from thermophilic Bacillus PS3 (TFoF1) and Escherichia coli, that the smallest subunits of F1, ϵ acts as an endogenous inhibitor of ATPase activity under some conditions (11Smith J.B. Sternweis P.C. Biochemistry. 1977; 16: 306-311Crossref PubMed Scopus (135) Google Scholar, 12Kato 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, 13Feniouk B.A. Suzuki T. Yoshida M. Biochim. Biophys. Acta. 2006; 1757: 326-338Crossref PubMed Scopus (82) Google Scholar, 14Feniouk B.A. Yoshida M. Results Probl. Cell Differ. 2008; 45: 279-308Crossref PubMed Scopus (45) Google Scholar). It is a 15-kDa protein composed of an N-terminal β-sandwich (∼80 residues) and C-terminal α-helical (∼50 residues) domains (15Uhlin U. Cox G.B. Guss J.M. Structure. 1997; 5: 1219-1230Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 16Wilkens S. Dahlquist F.W. McIntosh L.P. Donaldson L.W. Capaldi R.A. Nat. Struct. Biol. 1995; 2: 961-967Crossref PubMed Scopus (155) Google Scholar). Two α-helices in the C-terminal domain undergo large conformational transition between the “up-state,” in which the helices are extended as a long arm, and the C terminus reaches near the catalytic sites inside the α3β3-ring, and the “down-state,” in which the helices are retracted as a hairpin beside a globular domain of the γ-subunit (17Rodgers A.J. Wilce M.C. Nat. Struct. Biol. 2000; 7: 1051-1054Crossref PubMed Scopus (152) Google Scholar, 18Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 19Tsunoda S.P. Rodgers A.J. Aggeler R. Wilce M.C. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 6560-6564Crossref PubMed Scopus (160) Google Scholar). When ϵ is in the up-state, ATP hydrolysis is suppressed and transition to the down-state accompanies activation (20Suzuki 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 (127) Google Scholar, 21Iino 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 (62) Google Scholar, 22Saita E. Iino R. Suzuki T. Feniouk B.A. Kinosita Jr., K. Yoshida M. J. Biol. Chem. 2010; 285: 11411-11417Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The transition is induced by ATP (but not ADP) binding to F1, and then the down-state is further stabilized by binding of ATP directly to ϵ (21Iino 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 (62) Google Scholar, 23Kato-Yamada Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 24Yagi 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 (91) Google Scholar). Consequently, ϵ favors the up-state in the absence of ATP even when high concentration of ADP is present, and it undergoes the transition to the down-state as ATP concentration raises. Apparent binding affinities of ATP (Kd) to cause the transition is 140 μm for TFoF1 at 59 °C, a physiological temperature of Bacillus PS3 (25Feniouk B.A. Kato-Yamada Y. Yoshida M. Suzuki T. Biophys. J. 2010; 98: 434-442Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). These values obviously are lower than the normal cellular ATP concentration of bacteria (∼3 mm in E. coli) (26Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 1st Ed. American Society for Microbiology, Washington, D. C1987: 445-473Google Scholar). Therefore, the function of C-terminal helices is assumed as “emergency brake” to prevent ATP hydrolysis, which works only when cellular ATP concentration decreased drastically under severely starving conditions (25Feniouk B.A. Kato-Yamada Y. Yoshida M. Suzuki T. Biophys. J. 2010; 98: 434-442Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 27Feniouk B.A. Suzuki T. Yoshida M. J. Biol. Chem. 2007; 282: 764-772Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar).These contentions have been deduced mostly from analysis of ATP hydrolysis, and here, we studied the effect of conformational state of ϵ on ATP synthesis. As we have shown previously (20Suzuki 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 (127) Google Scholar, 21Iino 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 (62) Google Scholar), ϵ in TF1 is predominantly in the up-state when ATP in the solution is eliminated by hexokinase. As a control mimicking TFoF1 with the down-state ϵ, we used a mutant ϵΔc-TFoF1, in which ϵ lacks C-terminal helices (27Feniouk B.A. Suzuki T. Yoshida M. J. Biol. Chem. 2007; 282: 764-772Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 28Cipriano D.J. Dunn S.D. J. Biol. Chem. 2006; 281: 501-507Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 29Konno H. Suzuki T. Bald D. Yoshida M. Hisabori T. Biochem. Biophys. Res. Commun. 2004; 318: 17-24Crossref PubMed Scopus (12) Google Scholar, 30Masaike T. Suzuki T. Tsunoda S.P. Konno H. Yoshida M. Biochem. Biophys. Res. Commun. 2006; 342: 800-807Crossref PubMed Scopus (32) Google Scholar). Comparison of nucleotide specificity of wild-type TFoF1 (WT-TFoF1) and ϵΔc-TFoF1 in NTP (ATP, GTP, UTP, or CTP) synthesis reactions in the presence of hexokinase suggests that, when ϵ adopts the up-state conformation, TFoF1 gains higher nucleotide specificity to ATP. Results of NTP-driven proton pumping by ϵΔc-TFoF1 and NTP synthesis in the presence of adenosine 5′-(β,γ-imino)triphosphate (AMP-PNP) by TFoF1 are interpreted consistently by the above contention.DISCUSSIONThe ϵ-subunit has been long known as an intrinsic inhibitor of ATPase activity of F1 and FoF1. Recent studies revealed that ϵ can adopt two conformations, and the up-state conformation is responsible for the inhibition. Furthermore, it was found that ATP can bind to and stabilizes the down-state ϵ in some bacteria (13Feniouk B.A. Suzuki T. Yoshida M. Biochim. Biophys. Acta. 2006; 1757: 326-338Crossref PubMed Scopus (82) Google Scholar). Here, we added another novel aspect of the functions of ϵ, that is, modulation of nucleotide specificity. Given that AMP-PNP would stabilize the down-state ϵ in TFoF1 under the experimental conditions, we can indicate that the native TFoF1 of Bacillus PS3 might also modulate nucleotide specificity by ϵ at a physiological temperature (Fig. 4). As well, ϵ is likely to modulates nucleotide specificity in ATP hydrolysis (Fig. 5).From a structural aspect, higher nucleotide specificity of TFoF1 with the up-state ϵ is not surprising because the residues of the C-terminal region of ϵ can reach the location close to the catalytic site when C-terminal helices of the up-state ϵ are extended like a long arm into the inside of α3β3 ring. We think it is likely because the introduced cysteines at βLys334 near the catalytic site and ϵGlu131 at the third position from the C terminus can form a disulfide cross-link when ϵ is up-state in the absence of nucleotide. 3T. Suzuki, C. Wakabayashi, K. Tanaka, B. A. Feniouk, and M. Yoshida, unpublished observations. In the actively growing E. coli cell, cellular ATP concentration is in the range of ∼3 mm (26Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 1st Ed. American Society for Microbiology, Washington, D. C1987: 445-473Google Scholar) and concentrations of other three NTPs are estimated to be ∼1.5 mm or 0.7–1.2 mm (GTP), and ∼0.7 mm (UTP and CTP) (37Bagnara A.S. Finch L.R. Eur. J. Biochem. 1973; 36: 422-427Crossref PubMed Scopus (35) Google Scholar, 38Morikawa M. Izui K. Taguchi M. Katsuki H. J. Biochem. 1980; 87: 441-449Crossref PubMed Scopus (54) Google Scholar). Therefore, hydrolysis of the three NTPs by FoF1 with the down-state ϵ and, synthesis as well, can occur in the cell. Although its physiological significance is yet unclear, it is tempting to speculate that ϵ acts as an ATP-dependent switch that changes the mode of the enzyme operation. When the cell energy becomes poor and ATP concentration drops, TFoF1 would limit ATP and GTP consumption, and the synthesis activity is restricted to ATP.In summary, our results suggest that the ϵ-subunit of FoF1-ATP synthase from a thermophilic Bacillus regulates not only magnitude of activity but also modulates specificity of substrate nucleotide both in synthesis and hydrolysis reactions, depending on its extended and retracted conformations. IntroductionFoF1-ATP synthase (FoF1) 2The abbreviations used are: FoF1, FoF1-ATP synthase; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; CDTA, 1,2-cyclohexanediaminetetra-acetic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; TFoF1, FoF1-ATP synthase from thermophilic Bacillus PS3; NTP, four kinds nucleotide triphosphates (ATP, GTP, UTP, CTP); NDP, four kinds nucleotide diphosphates (ADP, GDP, UDP, CDP); IMV, inverted membrane vesicle. is ubiquitously found in membranes of bacteria, chloroplast, and mitochondria and synthesizes ATP by the energy of proton flow driven by the proton motive force. FoF1 also is able to catalyze the reverse reaction, ATP hydrolysis-driven proton pumping, which actually occurs in some cases and conditions. FoF1 is a motor enzyme composed of two rotary motors, membrane integral Fo, which converts the proton motive force into rotation, and water-soluble F1, which converts the rotation into synthesis of ATP (1Yoshida M. Muneyuki E. Hisabori T. Nat. Rev. Mol. Cell Biol. 2001; 2: 669-677Crossref PubMed Scopus (699) Google Scholar, 2Boyer P.D. J. Biol. Chem. 2002; 277: 39045-39061Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 3Senior A.E. Nadanaciva S. Weber J. Biochim. Biophys. Acta. 2002; 1553: 188-211Crossref PubMed Scopus (331) Google Scholar, 4Kinosita Jr., K. Adachi K. Itoh H. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 245-268Crossref PubMed Scopus (161) Google Scholar, 5Nakamoto R.K. Baylis Scanlon J.A. Al-Shawi M.K. Arch. Biochem. Biophys. 2008; 476: 43-50Crossref PubMed Scopus (127) Google Scholar, 6Hong S. Pedersen P.L. Microbiol. Mol. Biol. Rev. 2008; 72: 590-641Crossref PubMed Scopus (217) Google Scholar, 7Junge W. Sielaff H. Engelbrecht S. Nature. 2009; 459: 364-370Crossref PubMed Scopus (289) Google Scholar, 8Düser M.G. Zarrabi N. Cipriano D.J. Ernst S. Glick G.D. Dunn S.D. Börsch M. EMBO J. 2009; 28: 2689-2696Crossref PubMed Scopus (95) Google Scholar, 9von Ballmoos C. Wiedenmann A. Dimroth P. Annu. Rev. Biochem. 2009; 78: 649-672Crossref PubMed Scopus (261) Google Scholar, 10Nakanishi-Matsui M. Sekiya M. Nakamoto R.K. Futai M. Biochim. Biophys. Acta. 2010; 1797: 1343-1352Crossref PubMed Scopus (74) Google Scholar). F1 has a subunit composition of α3β3γδϵ and acts as ATPase when isolated. It has been known for the typical bacterial enzymes from thermophilic Bacillus PS3 (TFoF1) and Escherichia coli, that the smallest subunits of F1, ϵ acts as an endogenous inhibitor of ATPase activity under some conditions (11Smith J.B. Sternweis P.C. Biochemistry. 1977; 16: 306-311Crossref PubMed Scopus (135) Google Scholar, 12Kato 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, 13Feniouk B.A. Suzuki T. Yoshida M. Biochim. Biophys. Acta. 2006; 1757: 326-338Crossref PubMed Scopus (82) Google Scholar, 14Feniouk B.A. Yoshida M. Results Probl. Cell Differ. 2008; 45: 279-308Crossref PubMed Scopus (45) Google Scholar). It is a 15-kDa protein composed of an N-terminal β-sandwich (∼80 residues) and C-terminal α-helical (∼50 residues) domains (15Uhlin U. Cox G.B. Guss J.M. Structure. 1997; 5: 1219-1230Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 16Wilkens S. Dahlquist F.W. McIntosh L.P. Donaldson L.W. Capaldi R.A. Nat. Struct. Biol. 1995; 2: 961-967Crossref PubMed Scopus (155) Google Scholar). Two α-helices in the C-terminal domain undergo large conformational transition between the “up-state,” in which the helices are extended as a long arm, and the C terminus reaches near the catalytic sites inside the α3β3-ring, and the “down-state,” in which the helices are retracted as a hairpin beside a globular domain of the γ-subunit (17Rodgers A.J. Wilce M.C. Nat. Struct. Biol. 2000; 7: 1051-1054Crossref PubMed Scopus (152) Google Scholar, 18Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 19Tsunoda S.P. Rodgers A.J. Aggeler R. Wilce M.C. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 6560-6564Crossref PubMed Scopus (160) Google Scholar). When ϵ is in the up-state, ATP hydrolysis is suppressed and transition to the down-state accompanies activation (20Suzuki 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 (127) Google Scholar, 21Iino 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 (62) Google Scholar, 22Saita E. Iino R. Suzuki T. Feniouk B.A. Kinosita Jr., K. Yoshida M. J. Biol. Chem. 2010; 285: 11411-11417Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The transition is induced by ATP (but not ADP) binding to F1, and then the down-state is further stabilized by binding of ATP directly to ϵ (21Iino 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 (62) Google Scholar, 23Kato-Yamada Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 24Yagi 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 (91) Google Scholar). Consequently, ϵ favors the up-state in the absence of ATP even when high concentration of ADP is present, and it undergoes the transition to the down-state as ATP concentration raises. Apparent binding affinities of ATP (Kd) to cause the transition is 140 μm for TFoF1 at 59 °C, a physiological temperature of Bacillus PS3 (25Feniouk B.A. Kato-Yamada Y. Yoshida M. Suzuki T. Biophys. J. 2010; 98: 434-442Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). These values obviously are lower than the normal cellular ATP concentration of bacteria (∼3 mm in E. coli) (26Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 1st Ed. American Society for Microbiology, Washington, D. C1987: 445-473Google Scholar). Therefore, the function of C-terminal helices is assumed as “emergency brake” to prevent ATP hydrolysis, which works only when cellular ATP concentration decreased drastically under severely starving conditions (25Feniouk B.A. Kato-Yamada Y. Yoshida M. Suzuki T. Biophys. J. 2010; 98: 434-442Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 27Feniouk B.A. Suzuki T. Yoshida M. J. Biol. Chem. 2007; 282: 764-772Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar).These contentions have been deduced mostly from analysis of ATP hydrolysis, and here, we studied the effect of conformational state of ϵ on ATP synthesis. As we have shown previously (20Suzuki 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 (127) Google Scholar, 21Iino 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 (62) Google Scholar), ϵ in TF1 is predominantly in the up-state when ATP in the solution is eliminated by hexokinase. As a control mimicking TFoF1 with the down-state ϵ, we used a mutant ϵΔc-TFoF1, in which ϵ lacks C-terminal helices (27Feniouk B.A. Suzuki T. Yoshida M. J. Biol. Chem. 2007; 282: 764-772Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 28Cipriano D.J. Dunn S.D. J. Biol. Chem. 2006; 281: 501-507Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 29Konno H. Suzuki T. Bald D. Yoshida M. Hisabori T. Biochem. Biophys. Res. Commun. 2004; 318: 17-24Crossref PubMed Scopus (12) Google Scholar, 30Masaike T. Suzuki T. Tsunoda S.P. Konno H. Yoshida M. Biochem. Biophys. Res. Commun. 2006; 342: 800-807Crossref PubMed Scopus (32) Google Scholar). Comparison of nucleotide specificity of wild-type TFoF1 (WT-TFoF1) and ϵΔc-TFoF1 in NTP (ATP, GTP, UTP, or CTP) synthesis reactions in the presence of hexokinase suggests that, when ϵ adopts the up-state conformation, TFoF1 gains higher nucleotide specificity to ATP. Results of NTP-driven proton pumping by ϵΔc-TFoF1 and NTP synthesis in the presence of adenosine 5′-(β,γ-imino)triphosphate (AMP-PNP) by TFoF1 are interpreted consistently by the above contention. FoF1-ATP synthase (FoF1) 2The abbreviations used are: FoF1, FoF1-ATP synthase; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; CDTA, 1,2-cyclohexanediaminetetra-acetic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; TFoF1, FoF1-ATP synthase from thermophilic Bacillus PS3; NTP, four kinds nucleotide triphosphates (ATP, GTP, UTP, CTP); NDP, four kinds nucleotide diphosphates (ADP, GDP, UDP, CDP); IMV, inverted membrane vesicle. is ubiquitously found in membranes of bacteria, chloroplast, and mitochondria and synthesizes ATP by the energy of proton flow driven by the proton motive force. FoF1 also is able to catalyze the reverse reaction, ATP hydrolysis-driven proton pumping, which actually occurs in some cases and conditions. FoF1 is a motor enzyme composed of two rotary motors, membrane integral Fo, which converts the proton motive force into rotation, and water-soluble F1, which converts the rotation into synthesis of ATP (1Yoshida M. Muneyuki E. Hisabori T. Nat. Rev. Mol. Cell Biol. 2001; 2: 669-677Crossref PubMed Scopus (699) Google Scholar, 2Boyer P.D. J. Biol. Chem. 2002; 277: 39045-39061Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 3Senior A.E. Nadanaciva S. Weber J. Biochim. Biophys. Acta. 2002; 1553: 188-211Crossref PubMed Scopus (331) Google Scholar, 4Kinosita Jr., K. Adachi K. Itoh H. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 245-268Crossref PubMed Scopus (161) Google Scholar, 5Nakamoto R.K. Baylis Scanlon J.A. Al-Shawi M.K. Arch. Biochem. Biophys. 2008; 476: 43-50Crossref PubMed Scopus (127) Google Scholar, 6Hong S. Pedersen P.L. Microbiol. Mol. Biol. Rev. 2008; 72: 590-641Crossref PubMed Scopus (217) Google Scholar, 7Junge W. Sielaff H. Engelbrecht S. Nature. 2009; 459: 364-370Crossref PubMed Scopus (289) Google Scholar, 8Düser M.G. Zarrabi N. Cipriano D.J. Ernst S. Glick G.D. Dunn S.D. Börsch M. EMBO J. 2009; 28: 2689-2696Crossref PubMed Scopus (95) Google Scholar, 9von Ballmoos C. Wiedenmann A. Dimroth P. Annu. Rev. Biochem. 2009; 78: 649-672Crossref PubMed Scopus (261) Google Scholar, 10Nakanishi-Matsui M. Sekiya M. Nakamoto R.K. Futai M. Biochim. Biophys. Acta. 2010; 1797: 1343-1352Crossref PubMed Scopus (74) Google Scholar). F1 has a subunit composition of α3β3γδϵ and acts as ATPase when isolated. It has been known for the typical bacterial enzymes from thermophilic Bacillus PS3 (TFoF1) and Escherichia coli, that the smallest subunits of F1, ϵ acts as an endogenous inhibitor of ATPase activity under some conditions (11Smith J.B. Sternweis P.C. Biochemistry. 1977; 16: 306-311Crossref PubMed Scopus (135) Google Scholar, 12Kato 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, 13Feniouk B.A. Suzuki T. Yoshida M. Biochim. Biophys. Acta. 2006; 1757: 326-338Crossref PubMed Scopus (82) Google Scholar, 14Feniouk B.A. Yoshida M. Results Probl. Cell Differ. 2008; 45: 279-308Crossref PubMed Scopus (45) Google Scholar). It is a 15-kDa protein composed of an N-terminal β-sandwich (∼80 residues) and C-terminal α-helical (∼50 residues) domains (15Uhlin U. Cox G.B. Guss J.M. Structure. 1997; 5: 1219-1230Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 16Wilkens S. Dahlquist F.W. McIntosh L.P. Donaldson L.W. Capaldi R.A. Nat. Struct. Biol. 1995; 2: 961-967Crossref PubMed Scopus (155) Google Scholar). Two α-helices in the C-terminal domain undergo large conformational transition between the “up-state,” in which the helices are extended as a long arm, and the C terminus reaches near the catalytic sites inside the α3β3-ring, and the “down-state,” in which the helices are retracted as a hairpin beside a globular domain of the γ-subunit (17Rodgers A.J. Wilce M.C. Nat. Struct. Biol. 2000; 7: 1051-1054Crossref PubMed Scopus (152) Google Scholar, 18Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 19Tsunoda S.P. Rodgers A.J. Aggeler R. Wilce M.C. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 6560-6564Crossref PubMed Scopus (160) Google Scholar). When ϵ is in the up-state, ATP hydrolysis is suppressed and transition to the down-state accompanies activation (20Suzuki 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 (127) Google Scholar, 21Iino 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 (62) Google Scholar, 22Saita E. Iino R. Suzuki T. Feniouk B.A. Kinosita Jr., K. Yoshida M. J. Biol. Chem. 2010; 285: 11411-11417Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The transition is induced by ATP (but not ADP) binding to F1, and then the down-state is further stabilized by binding of ATP directly to ϵ (21Iino 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 (62) Google Scholar, 23Kato-Yamada Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 24Yagi 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 (91) Google Scholar). Consequently, ϵ favors the up-state in the absence of ATP even when high concentration of ADP is present, and it undergoes the transition to the down-state as ATP concentration raises. Apparent binding affinities of ATP (Kd) to cause the transition is 140 μm for TFoF1 at 59 °C, a physiological temperature of Bacillus PS3 (25Feniouk B.A. Kato-Yamada Y. Yoshida M. Suzuki T. Biophys. J. 2010; 98: 434-442Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). These values obviously are lower than the normal cellular ATP concentration of bacteria (∼3 mm in E. coli) (26Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 1st Ed. American Society for Microbiology, Washington, D. C1987: 445-473Google Scholar). Therefore, the function of C-terminal helices is assumed as “emergency brake” to prevent ATP hydrolysis, which works only when cellular ATP concentration decreased drastically under severely starving conditions (25Feniouk B.A. Kato-Yamada Y. Yoshida M. Suzuki T. Biophys. J. 2010; 98: 434-442Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 27Feniouk B.A. Suzuki T. Yoshida M. J. Biol. Chem. 2007; 282: 764-772Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). These contentions have been deduced mostly from analysis of ATP hydrolysis, and here, we studied the effect of conformational state of ϵ on ATP synthesis. As we have shown previously (20Suzuki 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 (127) Google Scholar, 21Iino 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 (62) Google Scholar), ϵ in TF1 is predominantly in the up-state when ATP in the solution is eliminated by hexokinase. As a control mimicking TFoF1 with the down-state ϵ, we used a mutant ϵΔc-TFoF1, in which ϵ lacks C-terminal helices (27Feniouk B.A. Suzuki T. Yoshida M. J. Biol. Chem. 2007; 282: 764-772Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 28Cipriano D.J. Dunn S.D. J. Biol. Chem. 2006; 281: 501-507Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 29Konno H. Suzuki T. Bald D. Yoshida M. Hisabori T. Biochem. Biophys. Res. Commun. 2004; 318: 17-24Crossref PubMed Scopus (12) Google Scholar, 30Masaike T. Suzuki T. Tsunoda S.P. Konno H. Yoshida M. Biochem. Biophys. Res. Commun. 2006; 342: 800-807Crossref PubMed Scopus (32) Google Scholar). Comparison of nucleotide specificity of wild-type TFoF1 (WT-TFoF1) and ϵΔc-TFoF1 in NTP (ATP, GTP, UTP, or CTP) synthesis reactions in the presence of hexokinase suggests that, when ϵ adopts the up-state conformation, TFoF1 gains higher nucleotide specificity to ATP. Results of NTP-driven proton pumping by ϵΔc-TFoF1 and NTP synthesis in the presence of adenosine 5′-(β,γ-imino)triphosphate (AMP-PNP) by TFoF1 are interpreted consistently by the above contention. DISCUSSIONThe ϵ-subunit has been long known as an intrinsic inhibitor of ATPase activity of F1 and FoF1. Recent studies revealed that ϵ can adopt two conformations, and the up-state conformation is responsible for the inhibition. Furthermore, it was found that ATP can bind to and stabilizes the down-state ϵ in some bacteria (13Feniouk B.A. Suzuki T. Yoshida M. Biochim. Biophys. Acta. 2006; 1757: 326-338Crossref PubMed Scopus (82) Google Scholar). Here, we added another novel aspect of the functions of ϵ, that is, modulation of nucleotide specificity. Given that AMP-PNP would stabilize the down-state ϵ in TFoF1 under the experimental conditions, we can indicate that the native TFoF1 of Bacillus PS3 might also modulate nucleotide specificity by ϵ at a physiological temperature (Fig. 4). As well, ϵ is likely to modulates nucleotide specificity in ATP hydrolysis (Fig. 5).From a structural aspect, higher nucleotide specificity of TFoF1 with the up-state ϵ is not surprising because the residues of the C-terminal region of ϵ can reach the location close to the catalytic site when C-terminal helices of the up-state ϵ are extended like a long arm into the inside of α3β3 ring. We think it is likely because the introduced cysteines at βLys334 near the catalytic site and ϵGlu131 at the third position from the C terminus can form a disulfide cross-link when ϵ is up-state in the absence of nucleotide. 3T. Suzuki, C. Wakabayashi, K. Tanaka, B. A. Feniouk, and M. Yoshida, unpublished observations. In the actively growing E. coli cell, cellular ATP concentration is in the range of ∼3 mm (26Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 1st Ed. American Society for Microbiology, Washington, D. C1987: 445-473Google Scholar) and concentrations of other three NTPs are estimated to be ∼1.5 mm or 0.7–1.2 mm (GTP), and ∼0.7 mm (UTP and CTP) (37Bagnara A.S. Finch L.R. Eur. J. Biochem. 1973; 36: 422-427Crossref PubMed Scopus (35) Google Scholar, 38Morikawa M. Izui K. Taguchi M. Katsuki H. J. Biochem. 1980; 87: 441-449Crossref PubMed Scopus (54) Google Scholar). Therefore, hydrolysis of the three NTPs by FoF1 with the down-state ϵ and, synthesis as well, can occur in the cell. Although its physiological significance is yet unclear, it is tempting to speculate that ϵ acts as an ATP-dependent switch that changes the mode of the enzyme operation. When the cell energy becomes poor and ATP concentration drops, TFoF1 would limit ATP and GTP consumption, and the synthesis activity is restricted to ATP.In summary, our results suggest that the ϵ-subunit of FoF1-ATP synthase from a thermophilic Bacillus regulates not only magnitude of activity but also modulates specificity of substrate nucleotide both in synthesis and hydrolysis reactions, depending on its extended and retracted conformations. The ϵ-subunit has been long known as an intrinsic inhibitor of ATPase activity of F1 and FoF1. Recent studies revealed that ϵ can adopt two conformations, and the up-state conformation is responsible for the inhibition. Furthermore, it was found that ATP can bind to and stabilizes the down-state ϵ in some bacteria (13Feniouk B.A. Suzuki T. Yoshida M. Biochim. Biophys. Acta. 2006; 1757: 326-338Crossref PubMed Scopus (82) Google Scholar). Here, we added another novel aspect of the functions of ϵ, that is, modulation of nucleotide specificity. Given that AMP-PNP would stabilize the down-state ϵ in TFoF1 under the experimental conditions, we can indicate that the native TFoF1 of Bacillus PS3 might also modulate nucleotide specificity by ϵ at a physiological temperature (Fig. 4). As well, ϵ is likely to modulates nucleotide specificity in ATP hydrolysis (Fig. 5). From a structural aspect, higher nucleotide specificity of TFoF1 with the up-state ϵ is not surprising because the residues of the C-terminal region of ϵ can reach the location close to the catalytic site when C-terminal helices of the up-state ϵ are extended like a long arm into the inside of α3β3 ring. We think it is likely because the introduced cysteines at βLys334 near the catalytic site and ϵGlu131 at the third position from the C terminus can form a disulfide cross-link when ϵ is up-state in the absence of nucleotide. 3T. Suzuki, C. Wakabayashi, K. Tanaka, B. A. Feniouk, and M. Yoshida, unpublished observations. In the actively growing E. coli cell, cellular ATP concentration is in the range of ∼3 mm (26Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 1st Ed. American Society for Microbiology, Washington, D. C1987: 445-473Google Scholar) and concentrations of other three NTPs are estimated to be ∼1.5 mm or 0.7–1.2 mm (GTP), and ∼0.7 mm (UTP and CTP) (37Bagnara A.S. Finch L.R. Eur. J. Biochem. 1973; 36: 422-427Crossref PubMed Scopus (35) Google Scholar, 38Morikawa M. Izui K. Taguchi M. Katsuki H. J. Biochem. 1980; 87: 441-449Crossref PubMed Scopus (54) Google Scholar). Therefore, hydrolysis of the three NTPs by FoF1 with the down-state ϵ and, synthesis as well, can occur in the cell. Although its physiological significance is yet unclear, it is tempting to speculate that ϵ acts as an ATP-dependent switch that changes the mode of the enzyme operation. When the cell energy becomes poor and ATP concentration drops, TFoF1 would limit ATP and GTP consumption, and the synthesis activity is restricted to ATP. In summary, our results suggest that the ϵ-subunit of FoF1-ATP synthase from a thermophilic Bacillus regulates not only magnitude of activity but also modulates specificity of substrate nucleotide both in synthesis and hydrolysis reactions, depending on its extended and retracted conformations. We thank Drs. E. Saita, N. Taniguchi, and Y. Kato-Yamada for discussion. Supplementary Material Download .pdf (.17 MB) Help with pdf files Download .pdf (.17 MB) Help with pdf files" @default.
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W1963539648 title "Modulation of Nucleotide Specificity of Thermophilic FoF1-ATP Synthase by ϵ-Subunit" @default.
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