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The N-terminal region of the ε subunit from cyanobacterial ATP synthase alone can inhibit ATPase activity

  • Kosuke Inabe
    Affiliations
    From the Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Nagatsuta-cho 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan

    School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho 4259, Midori-ku, Yokohama 226-8501, Japan
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  • Kumiko Kondo
    Affiliations
    From the Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Nagatsuta-cho 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan
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  • Keisuke Yoshida
    Affiliations
    From the Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Nagatsuta-cho 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan
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  • Ken-ichi Wakabayashi
    Affiliations
    From the Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Nagatsuta-cho 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan

    School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho 4259, Midori-ku, Yokohama 226-8501, Japan
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  • Toru Hisabori
    Correspondence
    To whom correspondence should be addressed:Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Nagatsuta-cho 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan
    Affiliations
    From the Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Nagatsuta-cho 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan

    School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho 4259, Midori-ku, Yokohama 226-8501, Japan
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  • Author Footnotes
    2 The abbreviations used are: CTDC-terminal domainNTDN-terminal domainAMS4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate.
Open AccessPublished:May 08, 2019DOI:https://doi.org/10.1074/jbc.RA118.007131
      ATP hydrolysis activity catalyzed by chloroplast and proteobacterial ATP synthase is inhibited by their ε subunits. To clarify the function of the ε subunit from phototrophs, here we analyzed the ε subunit–mediated inhibition (ε-inhibition) of cyanobacterial F1-ATPase, a subcomplex of ATP synthase obtained from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. We generated three C-terminal α-helix null ε-mutants; one lacked the C-terminal α-helices, and in the other two, the C-terminal conformation could be locked by a disulfide bond formed between two α-helices or an α-helix and a β-sandwich structure. All of these ε-mutants maintained ATPase-inhibiting competency. We then used single-molecule observation techniques to analyze the rotary motion of F1-ATPase in the presence of these ε-mutants. The stop angular position of the γ subunit in the presence of the ε-mutant was identical to that in the presence of the WT ε. Using magnetic tweezers, we examined recovery from the inhibited rotation and observed restoration of rotation by 80° forcing of the γ subunit in the case of the ADP-inhibited form, but not when the rotation was inhibited by the ε-mutants or by the WT ε subunit. These results imply that the C-terminal α-helix domain of the ε subunit of cyanobacterial enzyme does not directly inhibit ATP hydrolysis and that its N-terminal domain alone can inhibit the hydrolysis activity. Notably, this property differed from that of the proteobacterial ε, which could not tightly inhibit rotation. We conclude that phototrophs and heterotrophs differ in the ε subunit–mediated regulation of ATP synthase.

      Introduction

      ATP synthase synthesizes ATP from ADP and Pi using membrane potential, which is generated as a proton gradient across the bacterial plasma membrane, mitochondrial inner membrane, or chloroplast thylakoid membrane (
      • Yoshida M.
      • Muneyuki E.
      • Hisabori T.
      ATP synthase–a marvellous rotary engine of the cell.
      ,
      • Senior A.E.
      • Nadanaciva S.
      • Weber J.
      The molecular mechanism of ATP synthesis by F1F0-ATP synthase.
      • Mukherjee S.
      • Warshel A.
      The FOF1 ATP synthase: from atomistic three-dimensional structure to the rotary-chemical function.
      ). ATP synthase is composed of the hydrophilic portion F1 and the membrane-embedded hydrophobic portion Fo. The subunit composition of F1 is α3β3γδε (
      • Yoshida M.
      • Sone N.
      • Hirata H.
      • Kagawa Y.
      • Ui N.
      Subunit structure of adenosine triphosphatase: comparison of the structure in thermophilic bacterium PS3 with those in mitochondria, chloroplasts, and Escherichia coli.
      ), and that of Fo is ab2(or bb′)c10–15 in bacteria and chloroplasts (
      • Jiang W.
      • Hermolin J.
      • Fillingame R.H.
      The preferred stoichiometry of c subunits in the rotary motor sector of Escherichia coli ATP synthase is 10.
      ,
      • Pogoryelov D.
      • Reichen C.
      • Klyszejko A.L.
      • Brunisholz R.
      • Muller D.J.
      • Dimroth P.
      • Meier T.
      The oligomeric state of c rings from cyanobacterial F-ATP synthases varies from 13 to 15.
      • Vollmar M.
      • Schlieper D.
      • Winn M.
      • Büchner C.
      • Groth G.
      Structure of the c14 rotor ring of the proton translocating chloroplast ATP synthase.
      ). Fo of mitochondrial ATP synthase contains additional subunits, such as d, f, and h, which were not observed in those of bacterial and chloroplast-type ATP synthase (
      • Rühle T.
      • Leister D.
      Assembly of F1F0-ATP synthases.
      ). F1 is often referred to as F1-ATPase because F1 itself can catalyze the ATP hydrolysis reaction. This activity was thoroughly studied, and finally the rotary motion during the ATP hydrolysis reaction was directly visualized using single-molecule observation (
      • Noji H.
      • Yasuda R.
      • Yoshida M.
      • Kinosita Jr., K.
      Direct observation of the rotation of F1-ATPase.
      ,
      • Yasuda R.
      • Noji H.
      • Kinosita Jr., K.
      • Yoshida M.
      F1-ATPase is a highly efficient molecular motor that rotates with discrete 120 degree steps.
      ). In addition, the intrinsic regulation of this rotary motion caused by the ε subunit has also been reported in the case of thermophilic bacterial and cyanobacterial F1-ATPases (
      • Bald D.
      • Noji H.
      • Yoshida M.
      • Hirono-Hara Y.
      • Hisabori T.
      Redox regulation of the rotation of F1-ATP synthase.
      ,
      • Kim Y.
      • Konno H.
      • Sugano Y.
      • Hisabori T.
      Redox regulation of rotation of the cyanobacterial F1-ATPase containing thiol regulation switch.
      • Sunamura E.
      • Konno H.
      • Imashimizu M.
      • Mochimaru M.
      • Hisabori T.
      A conformational change of the γ subunit indirectly regulates the activity of cyanobacterial F1-ATPase.
      ).
      Because ATP synthase can potentially catalyze ATP hydrolysis when the membrane potential is insufficient for ATP synthesis, regulation of the activity should be important for living cells to avoid futile ATP hydrolysis reactions. One of the most well-known regulatory mechanisms is MgADP inhibition (ADP inhibition), which is induced by occupation of the catalytic site with MgADP and prevents the ATP hydrolysis reaction (
      • Vasilyeva E.A.
      • Fitin A.F.
      • Minkov I.B.
      • Vinogradov A.D.
      Kinetics of interaction of adenosine diphosphate and adenosine triphosphate with adenosine triphosphatase of bovine heart submitochondrial particles.
      ). The γ subunits of chloroplast-type ATP synthases, including the cyanobacterial one, possess an insertion region composed of 30–40 amino acids between the Rosmann-fold domain and the C-terminal domain (CTD)
      The abbreviations used are: CTD
      C-terminal domain
      NTD
      N-terminal domain
      AMS
      4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate.
      of α-helices, which is not observed in the γ subunit of other F1; this region has a function in regulating F1-ATPase (
      • Hisabori T.
      • Sunamura E.
      • Kim Y.
      • Konno H.
      The chloroplast ATP synthase features the characteristic redox regulation machinery.
      ,
      • Murakami S.
      • Kondo K.
      • Katayama S.
      • Hara S.
      • Sunamura E.I.
      • Yamashita E.
      • Groth G.
      • Hisabori T.
      Structure of the γ-ε complex of cyanobacterial F1-ATPase reveals a suppression mechanism of the γ subunit on ATP hydrolysis in phototrophs.
      ). In addition, the γ subunit of the chloroplast ATP synthase has an additional nine-amino acid insertion containing a pair of Cys residues at this insertion region, and this Cys pair is key for the redox control by thioredoxin (
      • Schwarz O.
      • Schürmann P.
      • Strotmann H.
      Kinetics and thioredoxin specificity of thiol modulation of the chloroplast H+-ATPase.
      ,
      • Stumpp M.T.
      • Motohashi K.
      • Hisabori T.
      Chloroplast thioredoxin mutants without active-site cysteines facilitate the reduction of the regulatory disulphide bridge on the γ-subunit of chloroplast ATP synthase.
      ). Under light conditions, this pair of Cys, which forms a disulfide bond under dark conditions, is reduced by thioredoxin, and consequently the catalytic activity is accelerated (
      • Kim Y.
      • Konno H.
      • Sugano Y.
      • Hisabori T.
      Redox regulation of rotation of the cyanobacterial F1-ATPase containing thiol regulation switch.
      ,
      • Nalin C.M.
      • McCarty R.E.
      Role of a disulfide bond in the γ subunit in activation of the ATPase of chloroplast coupling factor 1.
      ,
      • Konno H.
      • Nakane T.
      • Yoshida M.
      • Ueoka-Nakanishi H.
      • Hara S.
      • Hisabori T.
      Thiol modulation of the chloroplast ATP synthase is dependent on the energization of thylakoid membranes.
      ).
      In addition, F1-ATPases from chloroplasts and proteobacteria adopt the ε subunit as an intrinsic inhibitor for the ATP hydrolysis reaction (
      • Laget P.P.
      • Smith J.B.
      Inhibitory properties of endogenous subunit ε in the Escherichia coli F1 ATPase.
      ,
      • Konno H.
      • Murakami-Fuse T.
      • Fujii F.
      • Koyama F.
      • Ueoka-Nakanishi H.
      • Pack C.G.
      • Kinjo M.
      • Hisabori T.
      The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1-ATPase by the ε subunit.
      ). The ε subunit is composed of two domains, the N-terminal domain (NTD) with a β-sandwich structure and the CTD containing two tandem α-helices (
      • Uhlin U.
      • Cox G.B.
      • Guss J.M.
      Crystal structure of the ε subunit of the proton-translocating ATP synthase from Escherichia coli.
      ,
      • Yagi H.
      • Kajiwara N.
      • Tanaka H.
      • Tsukihara T.
      • Kato-Yamada Y.
      • Yoshida M.
      • Akutsu H.
      Structures of the thermophilic F1-ATPase ε subunit suggesting ATP-regulated arm motion of its C-terminal domain in F1.
      • Yagi H.
      • Konno H.
      • Murakami-Fuse T.
      • Isu A.
      • Oroguchi T.
      • Akutsu H.
      • Ikeguchi M.
      • Hisabori T.
      Structural and functional analysis of the intrinsic inhibitor subunit ε of F1-ATPase from photosynthetic organisms.
      ). The inhibitory mechanism by the ε subunit (ε-inhibition) has been proposed as follows: the ε subunit changes its conformation of two C-terminal α-helices from the retracted to the extended form in the enzyme complex in a manner dependent on a change in the microenvironment (
      • Suzuki T.
      • Murakami T.
      • Iino R.
      • Suzuki J.
      • Ono S.
      • Shirakihara Y.
      • Yoshida M.
      F0F1-ATPase/synthase is geared to the synthesis mode by conformational rearrangement of ε subunit in response to proton motive force and ADP/ATP balance.
      ). In the case of the bacterial ε subunit, this conformational change is caused by the binding and release of the ATP molecule at the CTD of α-helices (
      • Kato-Yamada Y.
      • Yoshida M.
      Isolated ε subunit of thermophilic F1-ATPase binds ATP.
      ) and/or the change in membrane potential (
      • Johnson E.A.
      • McCarty R.E.
      The carboxyl terminus of the epsilon subunit of the chloroplast ATP synthase is exposed during illumination.
      ). The extended CTD of α-helices of the ε subunit is then inserted into the cavity between the α and β subunits. This conformational change of the ε subunit enables the interaction between the positively charged CTD of the ε subunit and the negatively charged DELSEED motif of the β subunit (
      • Hara K.Y.
      • Kato-Yamada Y.
      • Kikuchi Y.
      • Hisabori T.
      • Yoshida M.
      The role of the betaDELSEED motif of F1-ATPase: propagation of the inhibitory effect of the ε subunit.
      ). This electrostatic interaction appeared to be the cause of inhibition of the enzyme because the deletion of the positively charged amino acid residues or the truncation of the CTD α-helices of the ε subunit diminished the inhibitory effect (
      • Hara K.Y.
      • Kato-Yamada Y.
      • Kikuchi Y.
      • Hisabori T.
      • Yoshida M.
      The role of the betaDELSEED motif of F1-ATPase: propagation of the inhibitory effect of the ε subunit.
      ,
      • Nowak K.F.
      • Tabidze V.
      • McCarty R.E.
      The C-terminal domain of the ε subunit of the chloroplast ATP synthase is not required for ATP synthesis.
      ). In contrast, the cyanobacterial ε subunit mutant exhibited different behavior. Truncation of the CTD containing α-helices of the ε subunit of cyanobacterial ATP synthase resulted in the decrease of ATP synthesis activity (
      • Imashimizu M.
      • Bernat G.
      • Sunamura E.
      • Broekmans M.
      • Konno H.
      • Isato K.
      • Rögner M.
      • Hisabori T.
      Regulation of F0F1-ATPase from Synechocystis sp. PCC 6803 by γ and ε subunits is significant for light/dark adaptation.
      ), whereas truncation to the same extent of the CTD containing α-helices of the ε subunit from proteobacteria resulted in an increase of ATP synthesis activity (
      • Masaike T.
      • Suzuki T.
      • Tsunoda S.P.
      • Konno H.
      • Yoshida M.
      Probing conformations of the β subunit of F0F1-ATP synthase in catalysis.
      ,
      • Iino R.
      • Hasegawa R.
      • Tabata K.V.
      • Noji H.
      Mechanism of inhibition by C-terminal α-helices of the ε subunit of Escherichia coli FOF1-ATP synthase.
      ). This implies that the ε subunit of cyanobacterial ATP synthase may have a different inhibitory mechanism from those in other organisms. In this study, to reveal the function of the ε subunit from cyanobacteria, three C-terminal α-helix null ε subunit mutants (CTD null ε-mutants) were constructed; the mutant lacking the C-terminal part containing α-helices (εN), the mutant whose C-terminal conformation can be locked by a disulfide bond formed between two α-helices (εCC_SS), and the other one whose C-terminal conformation can be locked by a disulfide bond formed between α-helix in the CTD and β-sandwich structure in the N-terminal domain (NTD) (εNC_SS). The inhibitory properties of these mutant ε subunits were thoroughly examined.

      Results

      ε-Mutants at the C-terminal domain

      To study the molecular mechanism of the ε-inhibition in cyanobacterial ATP synthase, three ε subunit mutants were prepared: εN, which consists of 83 amino acid residues at the NTD and lacks the CTD part containing α-helices; εCC_SS, whose Ala99 and Phe122 were substituted to Cys to allow disulfide bond formation between two α-helices (Fig. 1A); and εNC_SS, whose Thr46 and Arg124 were substituted with Cys to allow disulfide bond formation between the β-sandwich structure in the NTD and the α-helix in the CTD. We then determined the oxidant concentration that is sufficient for disulfide bond formation in εCC_SS and εNC_SS, and the reductant concentration to completely reduce these disulfide bonds (Fig. 1B). In the presence of more than 300 μm aldrithiol-2, disulfide bonds were formed between the two α-helices of εCC_SS, and between the β-sandwich structure in the NTD and the α-helix in the CTD of εNC_SS. The disulfide bond in εCC_SS was cleaved in the presence of 300 μm DTT. In contrast, the disulfide bond in εNC_SS was cleaved when 1 mm DTT was added. We therefore used 300 μm aldrithiol-2, and 300 μm or 1 mm DTT to control the disulfide bond formation in the mutants for further experiments. Hereafter, we describe εCC_SS _Ox as the oxidized εCC_SS, εCC_SS_Red as the reduced εCC_SS, and εCC_SS_Non as the untreated εCC_SS. In addition, εNC_SS_Ox as the oxidized εNC_SS, εNC_SS_Red as the reduced εNC_SS, and εNC_SS_Non as the untreated εNC_SS were used.
      Figure thumbnail gr1
      Figure 1The putative structure of the ε subunit mutants. A, structures of cyanobacterial ATP synthase ε subunit (εWT, PDB code 2RQ6) and the putative structure of mutants, εN, εCC_SS, and εNC_SS. B, the redox states of εCC_SS and εNC_SS. εCC_SS and εNC_SS was incubated in the presence of DTT or aldrithiol-2 with the indicated concentrations and then analyzed using nonreducing SDS-PAGE following AMS labeling. Red, reduced form; Ox, oxidized form.

      NTD of the ε subunit inhibits F1-ATPase activity

      Inhibition of the ATP hydrolysis activity of F1-ATPase was examined in the presence of the WT ε subunit (εWT) or its mutants (Fig. 2, A and B). The extent of the inhibition by εWT was very similar to that reported previously (
      • Konno H.
      • Murakami-Fuse T.
      • Fujii F.
      • Koyama F.
      • Ueoka-Nakanishi H.
      • Pack C.G.
      • Kinjo M.
      • Hisabori T.
      The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1-ATPase by the ε subunit.
      ). In contrast, one of our interesting findings is that εN clearly inhibited F1-ATPase activity even at 300 nm, which was comparable with the findings for εWT (Fig. 2A). This result was unexpected because CTD of the ε subunit was thought to be a key for the inhibition of F1-ATPase activity, as mentioned previously (
      • Johnson E.A.
      • McCarty R.E.
      The carboxyl terminus of the epsilon subunit of the chloroplast ATP synthase is exposed during illumination.
      ,
      • Hara K.Y.
      • Kato-Yamada Y.
      • Kikuchi Y.
      • Hisabori T.
      • Yoshida M.
      The role of the betaDELSEED motif of F1-ATPase: propagation of the inhibitory effect of the ε subunit.
      ), and to date no reports of the inhibition of F1-ATPase activity by NTD of the ε subunit have been published. In addition, we found that εCC_SS and εNC_SS can also inhibit F1-ATPase activity irrespective of their redox states (Fig. 2, B and C). These results also contradict the previous findings that the ε subunit mutant, which is incapable of conformational change at CTD, cannot inhibit the activity of F1-ATPase obtained from proteobacteria (
      • Shah N.B.
      • Hutcheon M.L.
      • Haarer B.K.
      • Duncan T.M.
      F1-ATPase of Escherichia coli: the ε-inhibited state forms after ATP hydrolysis, is distinct from the ADP-inhibited state, and responds dynamically to catalytic site ligands.
      ,
      • Kato-Yamada Y.
      • Yoshida M.
      • Hisabori T.
      Movement of the helical domain of the ε subunit is required for the activation of thermophilic F1-ATPase.
      ). The apparent dissociation constant (KD(app)) values between F1-ATPase and the ε subunit or its mutants were determined based on the ε-dependent decrease of ATP hydrolysis activity (Table 1). The KD(app) value for εWT was slightly lower than the previously reported value, 2.1 ± 0.3 nm (
      • Konno H.
      • Isu A.
      • Kim Y.
      • Murakami-Fuse T.
      • Sugano Y.
      • Hisabori T.
      Characterization of the relationship between ADP- and ε-induced inhibition in cyanobacterial F1-ATPase.
      ), but comparable with those in other reports (
      • Sunamura E.
      • Konno H.
      • Imashimizu M.
      • Mochimaru M.
      • Hisabori T.
      A conformational change of the γ subunit indirectly regulates the activity of cyanobacterial F1-ATPase.
      ,
      • Shah N.B.
      • Hutcheon M.L.
      • Haarer B.K.
      • Duncan T.M.
      F1-ATPase of Escherichia coli: the ε-inhibited state forms after ATP hydrolysis, is distinct from the ADP-inhibited state, and responds dynamically to catalytic site ligands.
      ). The KD(app) value for εN (21 ± 6.3 nm) was about 10 times greater than those for εWT and εCC_SS_Ox (2.5 ± 0.1 nm) and εNC_SS_Ox (2.3 ± 2.2 nm).
      Figure thumbnail gr2
      Figure 2Impacts of the ε-mutants on the F1-ATPase activity. A, ATPase activity of F1-ATPase in the presence of various concentrations of εWT or εN. Details of the assay conditions and the fitting equations are shown under “Experimental procedures.” For the ATPase assay, 2 nm F1-ATPase was used. Filled circles, εWT; open circles, εN. B, ATPase activity of F1-ATPase in the presence of various concentrations of εCC_SS. Filled squares, εCC_SS_Non; open square, εCC_SS_Ox; filled diamond, εCC_SS_Red. C, ATPase activity of F1-ATPase in the presence of various concentrations of εNC_SS. Open diamond, εNC_SS_Non; filled triangle, εNC_SS_Ox; open triangle, εNC_SS_Red. D, impacts of the ε-mutants on the F1-ATPase containing the γ subunit mutant (F1-ATPaseΔins). For the assay, 2 nm F1-ATPase was used. Filled bars, results of the WT F1-ATPase; open bars, results of the F1-ATPase mutant. 100 nm untreated (Non), oxidized (Ox), and reduced (Red) CTD null ε-mutants were used. All results shown in are the average of three independent experiments. Error bars, S.D.
      Table 1Apparent dissociation constants between F1-ATPase and εWT and mutants
      Type of εεWTεNεCC_SSεNC_SS
      NonRedOxNonRedOx
      KD(app) (nm)0.7 ± 0.321 ± 6.32.2 ± 0.52.8 ± 0.72.5 ± 0.11.1 ± 0.51.4 ± 0.72.3 ± 2.2
      Previously, we showed that the level of ε-inhibition of F1-ATPase containing the mutant γΔ198–222 subunit, which lacks the insertion region from Leu198 to Val222, apparently decreased to 20% (
      • Konno H.
      • Murakami-Fuse T.
      • Fujii F.
      • Koyama F.
      • Ueoka-Nakanishi H.
      • Pack C.G.
      • Kinjo M.
      • Hisabori T.
      The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1-ATPase by the ε subunit.
      ). We therefore applied this mutant ATPase complex (F1-ATPaseΔins) to investigate the inhibitory properties of εN. Neither εWT nor εN inhibited the ATP hydrolysis activity of the mutant ATPase complex (Fig. 2D), indicating that εN does not affect the activity of PK or LDH, which are used for our coupling assay system, and does not affect the α or β subunit of the complex as well.
      The KD(app) values of εSS under various oxidation or reduction conditions are shown in Table 1. No significant differences between the values that we obtained for εCC_SS or εNC_SS and those from the previous study on εWT were observed (
      • Konno H.
      • Isu A.
      • Kim Y.
      • Murakami-Fuse T.
      • Sugano Y.
      • Hisabori T.
      Characterization of the relationship between ADP- and ε-induced inhibition in cyanobacterial F1-ATPase.
      ).

      The properties of the ε-mutants

      To confirm that εN maintains the β-sandwich structure, we measured the CD spectrum of the protein. εN showed a negative peak at around 220 nm, which indicates the formation of the typical β-sheet (Fig. 3A) (
      • Kelly S.M.
      • Jess T.J.
      • Price N.C.
      How to study proteins by circular dichroism.
      ), and is different from the possible unfolded structure, because the latter shows a positive peak at 220 nm and negative peak at 200 nm (
      • Greenfield N.
      • Fasman G.D.
      Computed circular dichroism spectra for the evaluation of protein conformation.
      ). The CD spectra of εCC_SS suggested that the folding of εCC_SS is identical to that of εWT irrespective of its redox state (Fig. 3B). In addition, εCC_SS_Ox inhibited F1-ATPase activity, like εWT and εN (Fig. 2B). These results imply that the introduced Cys residues on the εCC_SS and the formation and dissociation of the disulfide bond in the CTD do not affect the affinity of εCC_SS to F1-ATPase. As shown in Table 1, KD(app) values of εN were weaker than those of εWT and the other mutants. To examine the binding of εN to F1-ATPase, we then tested the co-migration of F1-ATPase and εWT or εN by gel-filtration chromatography and analyzed the subunit composition in the peak by Western blotting (Fig. 4, A–D). When only F1-ATPase was subjected to the gel-filtration chromatography, a single peak was observed (Fig. 4A, peak 1). The β subunit was detected by Western blotting as indicated (Fig. 4D, lane 1). In contrast, two peaks were obtained when F1-ATPase was incubated with εWT or εN (Fig. 4B, peaks 2, 3, 5, and 6). After collecting these peak fractions, co-migrations of the ε subunits with F1-ATPase were examined by anti-ε subunit antibody (Fig. 4D, lanes 2, 3, 5, and 6). These protein bands showed the protein mass at around 15 and 10 kDa, which correspond to the molecular mass of εWT and εN, respectively. As a control, εN was subjected to gel-filtration chromatography (Fig. 4C, peak 7) solely, and the collected peak was analyzed by Western blotting (Fig. 4D, peak 7). Based on these results, we concluded that εN can directly bind to the F1-ATPase complex.
      Figure thumbnail gr3
      Figure 3Spectroscopic analysis of the ε-mutants structure. A, CD spectra of εWT or εN. Solid line, εWT; dotted line, εN. B, CD spectra of εCC_SS. Solid line, εCC_SS_Non; dotted line, εCC_SS_Ox; dashed line, εCC_SS_Red. The concentrations of the samples were 0.1 mg/ml.
      Figure thumbnail gr4
      Figure 4Complex formation between F1-ATPase and εWT or εN. Shown are gel-filtration chromatograms of F1-ATPase (α3β3γ subcomplex) (A), F1-ATPases incubated with εWT or εN (B), and εWT or εN (C). The peak was collected as an indicated number. D, the peak fractions corresponding to each number were collected, concentrated by TCA, and subjected to SDS-PAGE and analyzed by Western blotting using anti-β antibody and anti-ε antibody.

      Inhibition of F1-ATPase by εCC_SS at the single-molecule level

      To understand the molecular mechanism behind the inhibition by NTD of the ε subunit, the inhibition of rotation of the γ subunit by one of the CTD null ε-mutants εCC_SS_Ox was analyzed at the single-molecule level (Fig. 5). In this assay system, 3 μm εWT or εCC_SS_Ox was used. Although we examined 3 μm εN in this system, the marked change of rotation of the γ subunit was not observed. This must be due to the low affinity of εN to the γ subunit, which was lower than εWT or εCC_SS_Ox. We therefore tried to prepare 10 times higher concentrations of εN for this experiment. However, we failed to handle the higher concentration of εN in this study due to the low solubility of this mutant protein. Because the affinities of εCC_SS_Ox and εNC_SS_Ox to the γ subunit were similar (Table 1), we used εCC_SS_Ox for this rotation analysis. The rotary motion of the γ subunit was observed for 5 min under an optical microscope using 340-nm duplex polystyrene beads as a probe, followed by exchange of the assay buffer in the absence or presence of εWT or εCC_SS_Ox. As expected from the activity measurement (Fig. 2B), εWT and εCC_SS_Ox entirely stopped the rotation of F1-ATPase (Fig. 5, E and I). When a low concentration of ATP at 250 nm was used, the step of the γ subunit rotation at around 120° was observed (Fig. 5, B, F, and J). This step shows the ATP-waiting position when F1-ATPase is waiting for the next ATP binding (
      • Yasuda R.
      • Noji H.
      • Kinosita Jr., K.
      • Yoshida M.
      F1-ATPase is a highly efficient molecular motor that rotates with discrete 120 degree steps.
      ,
      • Konno H.
      • Murakami-Fuse T.
      • Fujii F.
      • Koyama F.
      • Ueoka-Nakanishi H.
      • Pack C.G.
      • Kinjo M.
      • Hisabori T.
      The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1-ATPase by the ε subunit.
      ). When εWT was infused into the observation chamber, rotation of the γ subunit stopped at an angular position that differed from the ATP-waiting position (Fig. 5, E–H), namely around 80° forward from that of the ATP-waiting position (Table 2). This observation was consistent with a previous report (
      • Konno H.
      • Murakami-Fuse T.
      • Fujii F.
      • Koyama F.
      • Ueoka-Nakanishi H.
      • Pack C.G.
      • Kinjo M.
      • Hisabori T.
      The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1-ATPase by the ε subunit.
      ). When εCC_SS_Ox was used, a similar cessation of rotation was observed (Fig. 5I), and the angular position was also around 80° (Fig. 5, I–L).
      Figure thumbnail gr5
      Figure 5Direct observation of the γ subunit rotation and its inhibition. Shown are total revolution numbers before and after the exchange of the assay buffer (A), those of εWT (E), and those of εCC_SS_Ox (I). The histograms of the stop angular position of the γ subunit before and after the exchange of assay buffer (B and C), those of εWT (F and G), and those of εCC_SS_Ox (J and K) were then calculated. The insets indicate the trajectories of each stop angular position of the γ subunit. D, H, and L, merged images of B and C, F and G, and J and K, respectively.
      Table 2Stop angular position of rotation of the γ subunit inhibited by the εWT or εCC_SS_Ox
      Type of εPausing angular position of γ (mean ± S.E.)
      −ε122 ± 4.3° (n = 12)
      WT86.1 ± 4.1° (n = 10)
      CC_SS Ox79.0 ± 3.1° (n = 12)

      Magnetic tweezer manipulation cannot recover the cessation of rotation by εCC_SS_Ox

      To distinguish the inhibition of F1-ATPase by ADP and that by ε, restoration of rotation of the γ subunit was thoroughly studied using the magnetic tweezer technique (
      • Konno H.
      • Isu A.
      • Kim Y.
      • Murakami-Fuse T.
      • Sugano Y.
      • Hisabori T.
      Characterization of the relationship between ADP- and ε-induced inhibition in cyanobacterial F1-ATPase.
      ,
      • Hirono-Hara Y.
      • Ishizuka K.
      • Kinosita Jr., K.
      • Yoshida M.
      • Noji H.
      Activation of pausing F1 motor by external force.
      ). For this purpose, a magnetic bead was attached to the γ subunit instead of the polystyrene beads, and the γ subunit stopped by the inhibition was forced 80° in the counterclockwise direction using the magnetic tweezers (Fig. 6A). ADP inhibition is a common way to inhibit F1-ATPase caused by the tightly bound ADP at the catalytic site(s) of the enzyme, and is conserved among the ATPases from mitochondria, proteobacteria, and chloroplasts. In the case of cyanobacterial F1-ATPase (
      • Konno H.
      • Isu A.
      • Kim Y.
      • Murakami-Fuse T.
      • Sugano Y.
      • Hisabori T.
      Characterization of the relationship between ADP- and ε-induced inhibition in cyanobacterial F1-ATPase.
      ) and thermophilic bacterial F1-ATPase (
      • Hirono-Hara Y.
      • Ishizuka K.
      • Kinosita Jr., K.
      • Yoshida M.
      • Noji H.
      Activation of pausing F1 motor by external force.
      ), restoration by 80° forcing was observed at the ADP inhibition state, whereas the ε-inhibition was not recovered by this procedure. We therefore applied this technique to the εCC_SS_Ox-inhibited F1-ATPase. This time, restoration of rotation was observed in ADP-inhibited F1-ATPase by 80° forcing (Fig. 6, B and C), and the frequency of restoration was about 70% (Table 3). This value is fairly similar to that reported previously (86% in Ref.
      • Konno H.
      • Isu A.
      • Kim Y.
      • Murakami-Fuse T.
      • Sugano Y.
      • Hisabori T.
      Characterization of the relationship between ADP- and ε-induced inhibition in cyanobacterial F1-ATPase.
      ). In contrast, εWT-inhibited F1-ATPase did not restore the rotation after 80° forcing (Fig. 6D). Restoration was also not observed in the case of εCC_SS_Ox-inhibited F1-ATPase (Fig. 6E). These results are summarized in Table 3 and suggest that the mechanism of inhibition of εCC_SS_Ox is identical to that of εWT.
      Figure thumbnail gr6
      Figure 6Molecular manipulation of the γ subunit in the ADP- or ε-inhibited F1-ATPase with magnetic tweezers. A, scheme of the mechanical manipulation of the γ subunit. ADP- or ε-inhibited γ subunit was forced by 80°, which was maintained for 5 s. B and C, time-course data of the angle of the γ subunit under the mechanical manipulation of the ADP-inhibited γ subunit. Black dots, angular position of the γ subunit. Blue dots, angular position of the ADP-inhibited γ subunit. Red dots, angular position of the γ subunit, which was manipulated by magnetic tweezers. D and E, time-course data of the angle of the γ subunit under the mechanical manipulation of the ε-inhibited γ subunit when rotation was inhibited by the infusion of εWT (D) or εCC_SS_Ox (E). After observation of the rotation of the γ subunit during 30 s, 3 μm εWT or εCC_SS_Ox was infused into the chamber. Black dots, angular position of the γ subunit before the injection of εWT or εCC_SS_Ox. Green dots show the angular position of the γ subunit that was inhibited by εWT or εCC_SS_Ox. Red points show the angular position of the γ subunit that was manipulated by magnetic tweezers.
      Table 3Frequency of mechanical restoration of the ADP-, εWT-, or εCC-SS_Ox-inhibited γ subunit
      Stall angleActivatedNot activatedFrequency of activation (%)
      80° (ADP inhibition)7370 (n = 10)
      80° (εWT inhibition)080 (n = 8)
      80° (εCC_SS_Ox inhibition)080 (n = 8)

      Discussion

      In this study, we aimed to clarify the mechanism of ε-inhibition of cyanobacterial ATP synthase in detail. First, we found that three CTD null mutants, εN, εCC_SS, and εNC_SS, inhibited F1-ATPase activity based on the enzymatic analysis (Fig. 2, A–C). This was unexpected because the significance of CTD of the ε subunit for the inhibition of ATP hydrolysis has already been reported (
      • Hara K.Y.
      • Kato-Yamada Y.
      • Kikuchi Y.
      • Hisabori T.
      • Yoshida M.
      The role of the betaDELSEED motif of F1-ATPase: propagation of the inhibitory effect of the ε subunit.
      ,
      • Nowak K.F.
      • Tabidze V.
      • McCarty R.E.
      The C-terminal domain of the ε subunit of the chloroplast ATP synthase is not required for ATP synthesis.
      ). Single-molecule observation revealed that εCC_SS_Ox stopped rotation of the γ subunit at around 80° (Fig. 5, J–L). The restoration of rotation of the γ subunit by forcing the γ subunit was not observed when it was inhibited by εCC_SS_Ox (Fig. 6E), although ADP-inhibited F1-ATPase was easily restored by 80° forcing. We therefore concluded that the εCC_SS-inhibitory mechanism was identical to that of εWT inhibition. This result indicates that ε-inhibition of cyanobacterial ATP synthase can occur irrespective of the conformation of CTD of the ε subunit, although CTD of the ε subunit is required for the tight binding to the γ subunit (Table 1). To our knowledge, all previous studies on the proteobacterial ε subunit indicated that CTD is indispensable for ε-inhibition (
      • Feniouk B.A.
      • Suzuki T.
      • Yoshida M.
      The role of subunit epsilon in the catalysis and regulation of FOF1-ATP synthase.
      ,
      • Krah A.
      • Kato-Yamada Y.
      • Takada S.
      The structural basis of a high affinity ATP binding epsilon subunit from a bacterial ATP synthase.
      • Sielaff H.
      • Duncan T.M.
      • Börsch M.
      The regulatory subunit ε in Escherichia coli FOF1-ATP synthase.
      ), and no reports have described that only NTD of the ε subunit inhibited the ATP hydrolysis activity of F1-ATPase or FoF1-ATPase.
      Cyanobacteria are believed to be the origin of chloroplasts, which were incorporated into ancestor cells during symbiosis. Consequently, many metabolic processes in chloroplasts are very similar to those in cyanobacteria. Therefore, the ε subunit of cyanobacterial ATP synthase may also have inhibitory mechanisms similar to those of chloroplast ATP synthase. However, Nowak et al. (
      • Nowak K.F.
      • Tabidze V.
      • McCarty R.E.
      The C-terminal domain of the ε subunit of the chloroplast ATP synthase is not required for ATP synthesis.
      ) reported that the mutant ε subunit of chloroplast ATP synthase from spinach, which lacks CTD, cannot inhibit F1-ATPase activity. In contrast, our results clearly indicate that εN inhibits the activity (Fig. 2), although the affinity to the complex was lower than that for εWT (Fig. 2 and Table 1). There is therefore a possibility that the ε subunit that lacks CTD did not sufficiently associate with the γ subunit of chloroplast ATP synthase in their experimental conditions (
      • Nowak K.F.
      • Tabidze V.
      • McCarty R.E.
      The C-terminal domain of the ε subunit of the chloroplast ATP synthase is not required for ATP synthesis.
      ) and did not inhibit F1-ATPase activity very well. Recently, the whole molecular structure of chloroplast ATP synthase was determined by cryo-EM (
      • Hahn A.
      • Vonck J.
      • Mills D.J.
      • Meier T.
      • Kühlbrandt W.
      Structure, mechanism, and regulation of the chloroplast ATP synthase.
      ), and our group also determined the X-ray crystal structure of the cyanobacterial γ–ε subcomplex (
      • Murakami S.
      • Kondo K.
      • Katayama S.
      • Hara S.
      • Sunamura E.I.
      • Yamashita E.
      • Groth G.
      • Hisabori T.
      Structure of the γ-ε complex of cyanobacterial F1-ATPase reveals a suppression mechanism of the γ subunit on ATP hydrolysis in phototrophs.
      ). In both structures, CTD of the ε subunit showed a retracted form, although CTDs of the ε subunits from Escherichia coli and Geobacillus stearothermophilus (formerly known as thermophilic Bacillus PS3) were extended in the crystal structures (
      • Shirakihara Y.
      • Shiratori A.
      • Tanikawa H.
      • Nakasako M.
      • Yoshida M.
      • Suzuki T.
      Structure of a thermophilic F1-ATPase inhibited by an ε-subunit: deeper insight into the ε-inhibition mechanism.
      ,
      • Cingolani G.
      • Duncan T.M.
      Structure of the ATP synthase catalytic complex (F1) from Escherichia coli in an autoinhibited conformation.
      ). Recently, the structure of F1-ATPase from Caldalkalibacillus thermarum was reported, and the ε subunit was found as a retracted form in this complex, whereas CTD of the ε subunit exerted the inhibitory effect on the ATPase activity (
      • Keis S.
      • Stocker A.
      • Dimroth P.
      • Cook G.M.
      Inhibition of ATP hydrolysis by thermoalkaliphilic F1FO-ATP synthase is controlled by the C terminus of the ε subunit.
      ,
      • Ferguson S.A.
      • Cook G.M.
      • Montgomery M.G.
      • Leslie A.G.
      • Walker J.E.
      Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum.
      ). This might be another example of inhibition by the retracted form CTD of the ε subunit.
      The γ subunits of chloroplast-type (cyanobacteria and chloroplast-type) ATP synthase equip the insertion region (Fig. 7), which also functions to inhibit ATP hydrolysis activity (
      • Murakami S.
      • Kondo K.
      • Katayama S.
      • Hara S.
      • Sunamura E.I.
      • Yamashita E.
      • Groth G.
      • Hisabori T.
      Structure of the γ-ε complex of cyanobacterial F1-ATPase reveals a suppression mechanism of the γ subunit on ATP hydrolysis in phototrophs.
      ). We therefore investigated the involvement of this region with the ε-inhibition of cyanobacterial ATP synthase in Fig. 2C. We then prepared a γ subunit mutant of F1-ATPase lacking the insertion region of the γ subunit (F1-ATPaseΔins). The inhibition of the ATP hydrolysis activity of F1-ATPaseΔins by the ε subunit and its mutants (Fig. 2D) was not remarkable compared with that of the WT F1-ATPase. In the crystal structure of the cyanobacterial γ–ε subcomplex, the β5 strand at the proximal end of ε-NTD appeared to form a mixed parallel β-sheet with the β-strand of the γ subunit to provide tight coupling between the γ and ε subunits (see Fig. 1C of Ref.
      • Murakami S.
      • Kondo K.
      • Katayama S.
      • Hara S.
      • Sunamura E.I.
      • Yamashita E.
      • Groth G.
      • Hisabori T.
      Structure of the γ-ε complex of cyanobacterial F1-ATPase reveals a suppression mechanism of the γ subunit on ATP hydrolysis in phototrophs.
      ). Therefore, both the insertion region of the γ subunit of cyanobacterial ATP synthase and ε-NTD must be involved in the regulation of ATP hydrolysis by the tight interaction between the γ and ε subunits. In addition, the ε subunit may affect the conformation of the insertion region of the γ subunit, which was found to be a β-hairpin structure (
      • Murakami S.
      • Kondo K.
      • Katayama S.
      • Hara S.
      • Sunamura E.I.
      • Yamashita E.
      • Groth G.
      • Hisabori T.
      Structure of the γ-ε complex of cyanobacterial F1-ATPase reveals a suppression mechanism of the γ subunit on ATP hydrolysis in phototrophs.
      ,
      • Hahn A.
      • Vonck J.
      • Mills D.J.
      • Meier T.
      • Kühlbrandt W.
      Structure, mechanism, and regulation of the chloroplast ATP synthase.
      ), to control the ATP hydrolysis activity in cyanobacteria and have an impact on the redox state of the γ subunit from chloroplast ATP synthase as well (
      • Richter M.L.
      • Snyder B.
      • McCarty R.E.
      • Hammes G.G.
      Binding stoichiometry and structural mapping of the ε polypeptide of chloroplast coupling factor 1.
      ).
      Figure thumbnail gr7
      Figure 7Comparison of the amino acid sequence of the γ subunit from various species. Partial amino acid sequence alignment of the γ subunits from various organisms was performed using the ESPript program (http://espript.ibcp.fr/ESPript/ESPript/index.php) (
      • Robert X.
      • Gouet P.
      Deciphering key features in protein structures with the new ENDscript server.
      ). (Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.) The indicated numbers are the amino acid locations of the γ subunit of T. elongatus BP-1. Red box, strictly conserved amino acids; red letters, closely related amino acids. Black line, to Leu198–Val222 of the T. elongatus BP-1 γ subunit. B. taurus, Bos taurus; Bacillus PS3, thermophilic Bacillus PS3 (G. stearothermophilus); S. oleracea, Spinacia oleracea; A. thaliana, Arabidopsis thaliana chloroplast-type 1; Synechocystis, Synechocystis sp. PCC6803; T. elongatus BP-1, Thermosynechococcus elongatus BP-1.
      Only from these in vitro analyses, we could not draw a definitive conclusion on whether CTD of the ε subunit of cyanobacterial ATP synthase can change the conformation in the FoF1 complex and exert the inhibitory effect on ATP hydrolysis. However, it should be noted that there are some organisms whose ε subunit of ATP synthase lacks CTD (
      • Sielaff H.
      • Duncan T.M.
      • Börsch M.
      The regulatory subunit ε in Escherichia coli FOF1-ATP synthase.
      ,
      • Xie D.L.
      • Lill H.
      • Hauska G.
      • Maeda M.
      • Futai M.
      • Nelson N.
      The atp2 operon of the green bacterium Chlorobium limicola.
      ). In the case of mitochondria, the homologous protein of the ε subunit is the δ subunit, although the δ subunit is not supposed to inhibit ATP hydrolysis activity (
      • Walker J.E.
      The ATP synthase: the understood, the uncertain and the unknown.
      ). In addition, the mitochondrial δ subunit cannot change conformation due to the covered structure of the ε subunit in the complex. Instead, IF1 acts as an ATP hydrolysis inhibitor protein in mitochondrial F1. IF1 binds to the interface between the α and β subunits of F1-ATPase, whereas the orientation for insertion into the interface differs from that of ε-CTD of F1 from E. coli or G. stearothermophilus (
      • Shirakihara Y.
      • Shiratori A.
      • Tanikawa H.
      • Nakasako M.
      • Yoshida M.
      • Suzuki T.
      Structure of a thermophilic F1-ATPase inhibited by an ε-subunit: deeper insight into the ε-inhibition mechanism.
      ,
      • Cingolani G.
      • Duncan T.M.
      Structure of the ATP synthase catalytic complex (F1) from Escherichia coli in an autoinhibited conformation.
      ,
      • Gledhill J.R.
      • Montgomery M.G.
      • Leslie A.G.
      • Walker J.E.
      How the regulatory protein, IF1, inhibits F1-ATPase from bovine mitochondria.
      ). In these bacterial F1s, the decrease of membrane potential or change in ATP concentration is suggested to induce the conformational change of ε-CTD and regulate the ε-inhibition (
      • Johnson E.A.
      • McCarty R.E.
      The carboxyl terminus of the epsilon subunit of the chloroplast ATP synthase is exposed during illumination.
      ,
      • Iino R.
      • Murakami T.
      • Iizuka S.
      • Kato-Yamada Y.
      • Suzuki T.
      • Yoshida M.
      Real-time monitoring of conformational dynamics of the ε subunit in F1-ATPase.
      ). Interestingly, both the bacterial ε subunit and the mitochondrial IF1 use the α-helix part to regulate F1-ATPase activity, whereas the binding positions in the complex are very different. In contrast, our study clearly shows that the cyanobacterial ε subunit does not require conformational change in the part of CTD containing α-helices for its inhibitory function, although we cannot rule out the possibility that the conformational change of CTD may occur in the FoF1 complex. Cyanobacterial ATP synthase works for both the photophosphorylation and the oxidative phosphorylation reaction, and this unique feature might be the origin of the unique regulatory system.

      Experimental procedures

      Materials

      Biotin-PEAC5-maleimide was purchased from Dojindo (Kumamoto, Japan). ATP, phosphoenolpyruvate, and BSA were obtained from Sigma. Pyruvate kinase, lactate dehydrogenase, and NADH were purchased from Roche Diagnostics (Basel, Switzerland). Other chemicals were of the highest grade commercially available.

      Protein preparation

      In this study, the α3β3γ subcomplex from a thermophilic cyanobacterium (T. elongatus BP-1) (
      • Konno H.
      • Isu A.
      • Kim Y.
      • Murakami-Fuse T.
      • Sugano Y.
      • Hisabori T.
      Characterization of the relationship between ADP- and ε-induced inhibition in cyanobacterial F1-ATPase.
      ) was used as a WT F1-ATPase. The expression and purification of the F1-ATPase complex were described previously (
      • Konno H.
      • Isu A.
      • Kim Y.
      • Murakami-Fuse T.
      • Sugano Y.
      • Hisabori T.
      Characterization of the relationship between ADP- and ε-induced inhibition in cyanobacterial F1-ATPase.
      ). εN was generated by the PrimeSTAR Mutagenesis Basal kit (Takara, Shiga, Japan) using the mutation primers shown in Table 4, and εSS was generated by an infusion method (Takara) using the mutation primers shown in Table 4.
      Table 4Sequence of primers used in this study to generate the ε subunit mutants
      ε mutationSequence of mutation primers
      εN5′-CGAGCGCTAGAAGCTTGCGGCCGCA-3′
      5′-GCTTCTAGCGCTCGGCACCGTTGAC-3′
      εA99C5′-GCGGAGTTTGCCGCCTGTCAGGCTGCCCTCGCTC-3′
      5′-CGTCGCGCCCGTGCTCGCTTG-3′
      εF122C5′-GGCGGCAAACTCCGCCTTGGCTT-3′
      5′-AGCACGGGCGCGACGACAGGCTTGGGTGGCTTGAA-3′
      εT46C5′-TTAACTGCCTTGGAATGTGGTGTGATGCGGGTGCG-3′
      5′-TTCCAAGGCAGTTAAGAGGGGGGCA-3′
      εR124C5′-GCGCCCGTGCTCGCTTGCAGG-3′
      5′-CAAGCGAGCACGGGCGCAACGAAAGGCTTGGGTG-3′
      The ε subunit and its mutants were expressed in E. coli and purified as described previously (
      • Konno H.
      • Murakami-Fuse T.
      • Fujii F.
      • Koyama F.
      • Ueoka-Nakanishi H.
      • Pack C.G.
      • Kinjo M.
      • Hisabori T.
      The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1-ATPase by the ε subunit.
      ) with some modification. For purification of the ε subunits, the combination of anion-exchange chromatography using DEAE Sephacel (GE Healthcare) and hydrophobic interaction chromatography using a Phenyl-Toyopearl column (Tosoh, Tokyo, Japan) was used. For further purification, size-exclusion chromatography using a Superdex 75 column (GE Healthcare) equilibrated with 50 mm HEPES-KOH (pH 8.0) and 100 mm KCl (Buffer A) was performed. All proteins were stored at −80 °C until use.

      Oxidation or reduction of εSS

      εCC_SS or εNC_SS mutant was incubated with various concentrations of Aldrithiol-2 or DTT in Buffer A for 60 min at room temperature to obtain the oxidized form εCC_SS or εNC_SSCC_SS_Ox or εNC_SS_Ox) or the reduced form εCC_SS or εNC_SSCC_SS_Red or εNC_SS_Red). To remove oxidants or reductants, the protein solution was loaded onto a Microcon column (10-kDa cut-off; Millipore) and centrifuged repeatedly. To confirm the oxidation and reduction state of εSS, the mutant proteins were precipitated by adding 5% (w/v, final concentration) TCA. After centrifugation, the supernatant was removed, and the remaining oxidant was washed away with 500 μl of acetone. After the removal of acetone by centrifugation, the pellet was dissolved in 50 mm Tris-HCl (pH 8.0), 1% (w/v) SDS, and thiol-modifying reagent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate (AMS). After labeling for 1 h at room temperature, protein samples were subjected to nonreducing SDS-PAGE, and the redox state of εCC_SS or εNC_SS was confirmed by determining the mobility in the gel.

      ATP hydrolysis activity assay

      ATP hydrolysis activity was measured in the presence of an ATP-regenerating system in 50 mm HEPES-KOH (pH 8.0), 100 mm KCl, 2 mm MgCl2, 2 mm ATP, 50 μg/ml PK, 50 μg/ml LDH, 2 mm phosphoenolpyruvate, and 0.2 mm NADH (
      • Stiggall D.L.
      • Galante Y.M.
      • Hatefi Y.
      Preparation and properties of complex V.
      ). The assay was carried out at 25 °C. The ATP hydrolysis rate after the addition of F1-ATPase was determined by monitoring the decrease in NADH absorption at 340 nm using a spectrophotometer, V-550 (Jasco, Tokyo, Japan). The results of three independent experiments were averaged.

      Estimation of ε subunit binding based on ATP hydrolysis activity assay

      The dissociation constant was estimated from the extent of ATP hydrolysis activity in the presence of the ε subunit or its mutants. The ATP hydrolysis activity was determined from the steady-state slope of ATP hydrolysis (
      • Konno H.
      • Murakami-Fuse T.
      • Fujii F.
      • Koyama F.
      • Ueoka-Nakanishi H.
      • Pack C.G.
      • Kinjo M.
      • Hisabori T.
      The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1-ATPase by the ε subunit.
      ). The change in the extent of inhibition depending on the ε-concentration was then fitted using the following equation,
      y=100(100Amin)·(KD(app)+[ε]+[F1])(KD(app)+[ε]+[F1])24[ε][F1]2[F1]
      (Eq. 1)


      where y is the residual activity of ATP hydrolysis in the presence of each concentration of the ε subunit, Amin is the minimum residual ATP hydrolysis activity, KD(app) is the dissociation constant between F1-ATPase and the ε subunit or its mutant, [ε] is the final concentration of the ε subunit, and [F1] is the final concentration of F1-ATPase.

      CD spectrum

      The ε subunit or its mutants were diluted in 20 mm Tris-HCl (pH 8.0), and their CD spectra were obtained using a spectrophotometer, J-820 (Jasco, Tokyo, Japan), at room temperature. The concentration of the ε subunit or its mutants was 0.1 mg/ml.

      Estimation of ε subunit binding based on gel filtration chromatography

      The amount of εN bound to F1-ATPase was estimated from the fraction of gel-filtration chromatography. 1 μm F1-ATPase and 5 μm εWT or εN were incubated at room temperature for 10 min in Buffer B (50 mm HEPES-KOH (pH 8.0), 100 mm KCl, 0.1 mm MgCl2, 0.1 mm ATP). Then the mixture was subjected to gel-filtration chromatography using a Superdex 200 increase column equilibrated with Buffer B, and the peaks were collected as indicated (peaks 1–7). The proteins in the peak fractions were then precipitated by 5% (w/v, final concentration) TCA. After centrifugation, the supernatant was removed, and the remaining protein pellet was washed away with 500 μl of acetone. After the removal of acetone by centrifugation, the pellet was dissolved in 50 mm Tris-HCl (pH 8.0), 1% (w/v) SDS and subjected to SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane and detected by anti-β or -ε subunit antibodies.

      Single-molecule observation

      Rotation assays were carried out as reported previously (
      • Konno H.
      • Murakami-Fuse T.
      • Fujii F.
      • Koyama F.
      • Ueoka-Nakanishi H.
      • Pack C.G.
      • Kinjo M.
      • Hisabori T.
      The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1-ATPase by the ε subunit.
      ) with some minor modifications. Streptavidin-coated beads with a diameter of 340 nm were used. Observation of rotation of the γ subunit was performed at room temperature. In general, solution exchange in the flow chamber took 1–2 min. The rotation was analyzed using custom software, Trans Viewer, prepared by Dr. Yusung Kim (
      • Kim Y.
      • Konno H.
      • Sugano Y.
      • Hisabori T.
      Redox regulation of rotation of the cyanobacterial F1-ATPase containing thiol regulation switch.
      ). Molecular manipulation using magnetic tweezers was performed as reported previously except for the magnetic beads (Sera-Mag Magnetic Streptavidin-Coated Particles, GE Healthcare) (
      • Konno H.
      • Isu A.
      • Kim Y.
      • Murakami-Fuse T.
      • Sugano Y.
      • Hisabori T.
      Characterization of the relationship between ADP- and ε-induced inhibition in cyanobacterial F1-ATPase.
      ).

      Author contributions

      K. I. and T. H. conceived the study, and K. I., K. K., and K. Y. performed the experiments. K. W. and T. H. supervised the research. K. I., K. K., K. Y., K. W., and T. H. discussed the data. K. I. and T. H. wrote the paper, and K. K., K. Y., and K. W. commented on the manuscript.

      Acknowledgments

      We thank the Center for Biological Resources and Informatics and the Suzukakedai Design and Manufacturing Division of Tokyo Institute of Technology for technical support.

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