Advertisement

Real-time Monitoring of Conformational Dynamics of the ϵ Subunit in F1-ATPase*

  • Ryota Iino
    Affiliations
    ATP System Project, ERATO, Japan Science and Technology Agency, Nagatsuta 5800-3, Yokohama 226-0026, Japan, Tokyo 171-8501, Japan

    Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503, Japan
    Search for articles by this author
  • Tomoe Murakami
    Affiliations
    ATP System Project, ERATO, Japan Science and Technology Agency, Nagatsuta 5800-3, Yokohama 226-0026, Japan, Tokyo 171-8501, Japan
    Search for articles by this author
  • Satoshi Iizuka
    Affiliations
    Department of Life Science, Tokyo 171-8501, Japan
    Search for articles by this author
  • Yasuyuki Kato-Yamada
    Affiliations
    Department of Life Science, Tokyo 171-8501, Japan

    Frontier Project Life's Adaptation Strategies to Environmental Changes, College of Science, Rikkyo (St. Paul's) University, Tokyo 171-8501, Japan
    Search for articles by this author
  • Toshiharu Suzuki
    Affiliations
    ATP System Project, ERATO, Japan Science and Technology Agency, Nagatsuta 5800-3, Yokohama 226-0026, Japan, Tokyo 171-8501, Japan

    Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503, Japan
    Search for articles by this author
  • Masasuke Yoshida
    Correspondence
    To whom correspondence should be addressed. Tel.: 81-45-924-5233; Fax: 81-45-924-5277;
    Affiliations
    ATP System Project, ERATO, Japan Science and Technology Agency, Nagatsuta 5800-3, Yokohama 226-0026, Japan, Tokyo 171-8501, Japan

    Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503, Japan
    Search for articles by this author
  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.
Open AccessPublished:October 03, 2005DOI:https://doi.org/10.1074/jbc.M506160200
      It has been proposed that C-terminal two α-helices of the ϵ subunit of F1-ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F1 from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the γ subunit and C terminus of the ϵ subunit, respectively. High FRET (extended form) of F1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5′-Adenyl-β,γ-imidodiphosphate, ADP + AlF4-, ADP + NaN3, and GTP also caused the retracted form, indicating that ATP binding to the catalytic β subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the ϵ subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65 °C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated ϵ subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65 °C reached to 0.67 mm, a consistent value to assume that the ϵ subunit acts as a sensor of ATP concentration in the cell.
      A rotary motor F1-ATPase (F1)
      The abbreviations used are: F1
      F1-ATPase
      F0F1
      F0F1-ATP synthase
      FRET
      fluorescence resonance energy transfer
      AMPPNP
      5′-adenyl-β,γ-imidodiphosphate.
      2The abbreviations used are: F1
      F1-ATPase
      F0F1
      F0F1-ATP synthase
      FRET
      fluorescence resonance energy transfer
      AMPPNP
      5′-adenyl-β,γ-imidodiphosphate.
      is a water-soluble portion of F0F1-ATP synthase, which catalyzes ATP synthesis/hydrolysis coupled with a transmembrane proton translocation (
      • Boyer P.D.
      ,
      • Yoshida M.
      • Muneyuki E.
      • Hisabori T.
      ). F1 has a subunit structure of α3β3γδϵ; α and β subunits have a non-catalytic and catalytic nucleotide binding sites, respectively; γ subunit rotates in the α3β3 ring; δ subunit connects the ring to the stator part of F0; and ϵ subunit rotates together with γ subunit as a body. The ϵ subunit (∼14 kDa) has a regulatory function and consists of N-terminal β-sandwitch and C-terminal two α-helices (
      • Uhlin U.
      • Cox G.B.
      • Guss J.M.
      ,
      • Wilkens S.
      • Capaldi R.A.
      ).
      Previous structural studies of F1 indicated two conformations of the ϵ subunit with different arrangement of the two α-helices, that is, retracted folded-hairpin form and partly extended form (Fig. 1, A and B) (
      • Gibbons C.
      • Montgomery M.G.
      • Leslie A.G.
      • Walker J.E.
      ,
      • Rodgers A.J.
      • Wilce M.C.
      ,
      • Hausrath A.C.
      • Capaldi R.A.
      • Matthews B.W.
      ,
      • Tsunoda S.P.
      • Rodgers A.J.
      • Aggeler R.
      • Wilce M.C.
      • Yoshida M.
      • Capaldi R.A.
      ). Cross-linking studies suggested the third conformation with fully extended α-helices
      Recently, Y. Shirakihara and M. Yoshida and T. Suzuki determined the crystal structure of F1 that has the ϵ subunit with fully extended α-helices (unpublished data).
      3Recently, Y. Shirakihara and M. Yoshida and T. Suzuki determined the crystal structure of F1 that has the ϵ subunit with fully extended α-helices (unpublished data).
      (Fig. 1C) (
      • Suzuki T.
      • Murakami T.
      • Iino R.
      • Suzuki J.
      • Ono S.
      • Shirakihara Y.
      • Yoshida M.
      ). Biochemical data have indicated that the ϵ subunit adopts the extended form in the absence of nucleotide or in the presence of ADP, in which ATPase activity is inhibited, and that ATP counteracts ADP by favoring the retracted form, which is a non-inhibitory conformation (
      • Suzuki T.
      • Murakami T.
      • Iino R.
      • Suzuki J.
      • Ono S.
      • Shirakihara Y.
      • Yoshida M.
      ). Thus, it appears that the regulatory function of the ϵ subunit is dependent on the drastic conformational transition that is affected by nucleotide and other factors. However, previous studies have not provided kinetic information on how these dynamic conformational transitions occur in the enzyme at work.
      Figure thumbnail gr1
      FIGURE 1Probing conformational dynamics of the ϵ subunit by FRET. Structure of the γ (silver) and ϵ subunit (green) of F1 from bovine mitochondria (
      • Gibbons C.
      • Montgomery M.G.
      • Leslie A.G.
      • Walker J.E.
      ) (A), E. coli (
      • Rodgers A.J.
      • Wilce M.C.
      ,
      • Hausrath A.C.
      • Capaldi R.A.
      • Matthews B.W.
      ) (B), and thermophilic Bacillus PS3 (C)3. One α and β subunits are also shown (yellow). The δ subunit of bovine mitochondrial F1 is equivalent to the ϵ subunit of bacterial F1 and is referred to as the ϵ here. Cy3 (donor, red circle) and Cy5 (acceptor, blue circle) were conjugated with the γS3C and ϵ134C of F1 derived from thermophilic Bacillus PS3, respectively. The distances between two cysteine residues were estimated to be 8 nm (A), 2 nm (B), and 1 nm (C). Since the typical Förester distance is ∼5 nm, a large change in the FRET efficiency according to the conformational change was expected.
      Fluorescence resonance energy transfer (FRET) is a powerful technique that enables us to probe conformational dynamics of biological molecular machines (
      • Stryer L.
      ). In this study, this technique was applied to the ϵ subunit in F1 from thermophilic Bacillus PS3. Real-time FRET monitoring revealed slow, reversible conformational change of the ϵ subunit, indicating that the ϵ subunit exerts a slow switch-like regulation. In addition, direct binding of ATP to the isolated ϵ subunit was probed by fluorescence change of the dye attached to the ϵ subunit. Submillimolar affinity of the ϵ subunit to ATP at the physiological temperature of Bacillus PS3 (65 °C) suggests a novel function of the ϵ subunit as an ATP concentration sensor in vivo.

      EXPERIMENTAL PROCEDURES

      Protein Preparation—A mutant of the α3β3γ subcomplex (αC193S, βHis-10 at N terminus, γS3C) and mutants of the ϵ subunit (134C or Q107C) of F1 from thermophilic Bacillus PS3 were purified as described (
      • Shimabukuro K.
      • Yasuda R.
      • Muneyuki E.
      • Hara K.Y.
      • Kinosita Jr., K.
      • Yoshida M.
      ,
      • Kato-Yamada Y.
      • Yoshida M.
      ). ATPase activity of reconstituted α3β3γϵ subcomplex (referred as F1 hereafter) was measured as described previously (
      • Suzuki T.
      • Murakami T.
      • Iino R.
      • Suzuki J.
      • Ono S.
      • Shirakihara Y.
      • Yoshida M.
      ). A molecular extinction coefficient of 154,000 at 280 nm was used for the determination of F1 concentration.
      Reconstitution of Fluorescent-labeled α3β3γϵ Complex—For FRET measurement, α3β3γ subcomplex and ϵ subunit (134C) were reacted with Cy3- and Cy5-maleimide (Amersham Biosciences, dye/protein molar ratio was 2) in PA5 buffer (50 mm HEPES-KOH, 5 mm MgCl2, 100 mm KCl, pH 7.5) for 30 min at 25 °C, respectively, and unreacted dyes were removed by gel filtration. Labeling ratio of dye to protein was ∼0.5 and >0.9 for α3β3γ(S3C-Cy3) and ϵ(134C-Cy5), respectively (judged by SDS-PAGE). For reconstitution of α3β3γ(S3C-Cy3) ϵ(134C-Cy5) subcomplex, α3β3γ(S3C-Cy3) and ϵ(134C-Cy5) were mixed (molar ratio of ϵ subunit to α3β3γ was 3) and incubated for 3 h at 25°C, and excess ϵ(134C-Cy5) was removed by ultrafiltration. Reconstituted F1 contained 0.66 mol of ADP and 0.26 mol of ATP/mol of F1 and retained 76% steady-state ATPase activity as compared with that of wild type (134 ± 4.0 s-1 measured at 50 °C). From this result we concluded that dye-labeled F1 used in this study was functional. Molecular extinction coefficients of 150,000 at 550 nm (Cy3) and 250,000 at 650 nm (Cy5) were used for determination of concentration.
      FRET Measurement—FRET from the donor to the acceptor in the F1 was monitored with a fluorescence spectrometer (FP-3000, Hitachi). Donor was selectively excited with the light of 532 nm (bandwidth was 3 nm). For time course measurements, fluorescence intensity of donor was measured at 565 nm (bandwidth was 10 nm). Measurements were carried out in PA5 buffer, and an ATP (GTP) regenerating system (5 mm phosphoenolpyruvate and 0.5 mg/ml pyruvate kinase) was added in some experiments as described. Contamination of ATP in the solutions of AMPPNP and GTP was less than 0.1% (estimated by high performance liquid chromatography). Steady-state fluorescence anisotropy of protein-conjugated Cy3 and Cy5 in the buffer PA5 was measured in a cuvette with a fluorescence spectrometer (FP-3000).
      Measurement of Affinity between Nucleotides and Isolated ϵ Subunit—The Q107C mutant of the ϵ subunit was reacted with Cy3-maleimide (dye/protein molar ratio was 2), and unreacted dyes were removed by gel filtration. The labeling ratio of dye to protein was >0.9. Nucleotide was added to ϵ(Q107C-Cy3) in PA5 buffer, and fluorescence intensity was measured with a fluorescence spectrometer (FP-3000). Cy3 was excited with the light of 532 nm (bandwidth was 3 nm), and fluorescence intensity was measured at 565 nm (bandwidth was 10 nm). For the measurement of ADP affinity, hexokinase (6.7 units/ml) and glucose (200 mm) were supplemented to the buffer, and contaminated ATP was converted to ADP.

      RESULTS

      Nucleotide-dependent, Reversible Conformational Change of the ϵ Subunit—In a fluorescence spectrum of α3β3γ(S3C-Cy3)ϵ(134C-Cy5) obtained by excitation of donor dye (Cy3) at 532 nm (Fig. 2A, blue solid line), the peak of donor (Cy3, around 570 nm) decreased, as compared with the spectrum of α3β3γ(S3C-Cy3) (blue dashed line). Concomitantly the peak of acceptor (Cy5, around 670 nm) increased, indicating FRET from donor to acceptor with high efficiency. FRET was further confirmed by recovery of donor fluorescence intensity after photobleaching of acceptor by excitation at 670 nm (data not shown). When ATP (1 mm) was added, although the spectra of α3β3γ(S3C-Cy3) (red dashed line) and ϵ(134C-Cy5) (red dotted line) did not change, intensities of donor and acceptor fluorescence of α3β3γ(S3C-Cy3)ϵ(34C-Cy5) (red solid line) were increased and decreased, respectively. This ATP-induced transition from high FRET to low FRET primarily reflects the conformational change of the ϵ subunit because the γ and ϵ subunits rotate together as a body and the γ subunit does not undergo large conformational transition during catalysis. Hereafter, α3β3γ(S3C-Cy3)ϵ(134C-Cy5) subcomplex was referred as F1 in this study and was used for FRET measurement.
      Figure thumbnail gr2
      FIGURE 2Nucleotide dependence of FRET. A, fluorescence spectra of α3β3γ(S3C-Cy3) (dashed lines), ϵ(134C-Cy5) (dotted lines), and α3β3γ(S3C-Cy3)ϵ(134C-Cy5) (solid lines) before (blue) and after (red) addition of 1 mm ATP. Measurements were carried out at 35 °C. AU, arbitrary unit. B, change in the donor fluorescence intensity after addition of various nucleotides. Nucleotides (1 mm) were added at time 0. In the case of ADP + NaN3, NaN3 (2 mm) was also added at time 0. In case of ADP + AlF4-, AlCl3 (1 mm) and KF (4 mm) were added at 300 s before the addition of ADP. All measurements were carried out at 50 °C. In A and B, an ATP regenerating system was not included. C, effect of GTP on the donor fluorescecne intensity. Measurements were carried out at 35 °C in the presence of an ATP (GTP) regenerating system. In all measurements in this figure, F1 concentration was 10 nm.
      Apparent FRET efficiency before and after addition of ATP, estimated from the spectra shown in Fig. 2A, was about 70 and 20%, respectively. It should be mentioned that the distance between donor and acceptor could not be estimated precisely from FRET efficiency in our experiments, since steady-state fluorescence anisotropy of Cy3 and Cy5 conjugated with γS3C and ϵ134C was high (>0.36). A high value of the anisotropy means that the mobility of the dye molecules conjugated with F1 is highly restricted, and the estimation of the distance with high reliability is difficult. However, considering that the typical Förester distance (the distance which exhibits 50% FRET efficiency) is around 5 nm (
      • Stryer L.
      ), high (70% efficiency) and low FRET (20%) states presumably represent the “extended” conformation (the distance between cysteine residues is 1∼2 nm) and the “retracted” (8 nm) conformation of the ϵ subunit, respectively (Fig. 1). Since it was difficult to distinguish between “partly extended” (Fig. 1B) and “fully extended” (Fig. 1C) conformations in this study, these two conformations were treated together and were referred as the extended conformation.
      Time courses of the change of FRET efficiency induced by nucleotides were monitored by the change of the donor fluorescence intensity. As mentioned, 1 mm ATP increased donor fluorescence intensity (Fig. 2B, red line). On the contrary, ADP (1 mm) did not cause the change of donor fluorescence (Fig. 2B, blue line). AMPPNP (1 mm), which can bind to F1 but is not hydrolyzed (
      • Abrahams J.P.
      • Leslie A.G.
      • Lutter R.
      • Walker J.E.
      ), also caused the increase in donor fluorescence, although the change was much slower than the case of ATP (Fig. 2B, light blue line). Therefore, ATP binding but not hydrolysis induces the conformational change from the extended form to the retracted form. ADP + NaN3, which induces and stabilizes so-called “ADP-Mg inhibited state
      ADP-Mg inhibited state: the catalytic turnover of ATP hydrolysis by F1 is interrupted by occasional stable trapping of ADP-Mg at the catalytic site(s). The binding of ATP to the α subunits facilitates the release of ADP-Mg from the affected catalytic sites, thereby recovering the ATP hydrolysis activity. This ADP-Mg inhibited state is different from the product inhibition and is observed for nearly all F1s and ATP synthases from various sources.
      ” of F1 (
      • Muneyuki E.
      • Makino M.
      • Kamata H.
      • Kagawa Y.
      • Yoshida M.
      • Hirata H.
      ), and ADP + AlF4-, a transitionstate substrate analogue of ATP hydrolysis (
      • Braig K.
      • Menz R.I.
      • Montgomery M.G.
      • Leslie A.G.
      • Walker J.E.
      ), also increased the donor fluorescence intensity (Fig. 2B, green and yellow lines, respectively). Furthermore, GTP also induced the conformational change (Fig. 2C). GTP is a substrate of F1 and supports its rotation (
      • Noji H.
      • Bald D.
      • Yasuda R.
      • Itoh H.
      • Yoshida M.
      • Kinosita Jr., K.
      ) but has very low affinity to the isolated ϵ subunit (see Fig. 5) (
      • Kato-Yamada Y.
      • Yoshida M.
      ).
      Figure thumbnail gr5
      FIGURE 5Affinity of ATP to the isolated ϵ subunit. A, increase in the fluorescence intensity of isolated ϵ(Q107C-Cy3) in the presence of ATP (red), ADP (green), and GTP (blue). Nucleotides were added at the points indicated by arrowheads. The concentration of nucleotides was gradually increased from 1 nm to 3 mm (ATP) or 6 mm (ADP and GTP). Measurements were carried out at 36 °C, and hexokinase (6.7 units/ml) and glucose (200 mm) were supplemented for measurement with ADP. Due to the ATP contamination, transient overshoots were observed in the trace of ADP. B, plot of the change in fluorescence intensity against ATP (circles), ADP (squares), and GTP (triangles) concentration. The data were fitted assuming the simple binding reaction, and the dissociation constants of ATP, ADP, and GTP were calculated to be 1.4, 130, and 1.8 mm, respectively. C, temperature dependence of the dissociation constant of ATP (circles). Dissociation constants of ADP (squares) and GTP (triangles) at 36 °C are also shown. D, logarithmic plot of dissociation constant in micromolar against reciprocal temperature. The dissociation constant of ATP extrapolated to the physiological temperature (65 °C, dashed line) for Bacillus PS3 was 0.67 mm. The concentration of the ϵ subunit was 20 nm.
      The conformational change of the ϵ subunit was reversible. When added ATP in the medium was rapidly converted into ADP by addition of excess amount of hexokinase (27 units/ml), the change was reverted (Fig. 4B). These results provide direct evidence for nucleotide-dependent reversible conformational rearrangement of the ϵ subunit that was previously suggested from biochemical works (
      • Suzuki T.
      • Murakami T.
      • Iino R.
      • Suzuki J.
      • Ono S.
      • Shirakihara Y.
      • Yoshida M.
      ).
      Figure thumbnail gr4
      FIGURE 4Temperature dependence of the rate of conformational change. A, transition from the extended form to the retracted form induced by 1 mm ATP. Measurements were carried out in the presence of an ATP regenerating system. B, transition from the retracted form to the extended form. After transition to the retracted form induced by ATP (0.06 mm), ATP was converted into ADP by addition of hexokinase (27 units/ml). The transition rate increased with the amount of hexokinase but was saturated at 13 units/ml, indicating that ATP conversion into ADP by hexokinase was much faster than those of conformational change of the ϵ subunit. Glucose (200 mm) was supplemented to the buffer. C, temperature dependence of rate constant of conformational transition. Time courses were fitted as described in the legend to , and fast (triangles) and slow (inverted triangles) components of the extended-to-retracted (filled symbols) and retracted-to-extended (open symbols) transitions were plotted. The dashed line indicates physiological temperature (65 °C) of thermophilic Bacillus PS3. D, Arrhenius plot of rate constant. The fast components of rate constant of the extended-to-retracted and the retracted-to-extended transitions extrapolated to the physiological temperature (dashed line) were 2.7 and 0.18 s-1, respectively. F1 concentration was 10 nm.
      Rates of Recovery from Inhibition and Conformational Change—ATPase activity of F1 from thermophilic Bacillus PS3 is inhibited by the ϵ subunit but the inhibition is relieved slowly by addition of ATP (
      • Kato Y.
      • Matsui T.
      • Tanaka N.
      • Muneyuki E.
      • Hisabori T.
      • Yoshida M.
      ). Since it is thought that the extended ϵ subunit has inhibitory effect on ATPase activity of F1 but the retracted ϵ subunit does not (
      • Tsunoda S.P.
      • Rodgers A.J.
      • Aggeler R.
      • Wilce M.C.
      • Yoshida M.
      • Capaldi R.A.
      ,
      • Suzuki T.
      • Murakami T.
      • Iino R.
      • Suzuki J.
      • Ono S.
      • Shirakihara Y.
      • Yoshida M.
      ), recovery of ATPase activity from inhibition should accompany increase of donor fluorescence and we compared their time courses. ATPase activity of F1 is gradually recovered after initiation of the assay by addition of ATP (Fig. 3A) (
      • Kato Y.
      • Matsui T.
      • Tanaka N.
      • Muneyuki E.
      • Hisabori T.
      • Yoshida M.
      ). As ATP concentration increased, recovery occurred more rapidly. Time courses of the recovery could be fitted by a single exponential function assuming transition between the active and inactive states (Fig. 3C, inset, green line). Apparent rate constants estimated from the fitting became larger as ATP concentration increased (Fig. 3D, circles).
      Figure thumbnail gr3
      FIGURE 3Comparison between the rate of ATPase recovery of F1 and that of conformational change of the ϵ. A and B, increase in the ATPase activity (A) and increase in the donor fluorescence intensity (B) after the addition of ATP. ATP concentrations are 0.1 (blue), 0.3 (green),1(yellow), and 3 mm (red), respectively. In A and B, measurements were carried out in the presence of an ATP regenerating system at 35 °C. F1 concentration was 5 nm. C, the time courses shown in A and B (1 mm ATP) were fitted by a single exponential function assuming the reaction between two forms (green) or a following double exponential function (red): a + b[exp(-kfastt)] + c[exp(-kslowt)], where a, b, and c are constants, and kfast and kslow are fast and slow components of the rate constant. D, rate constant plotted against ATP concentration. Rate constants of increase in the ATPase activity (circles) and fast (triangles) and slow (inverted triangles) components of the rate constant of increase in donor fluorescence were plotted.
      The increase in donor fluorescence occurred in a time scale similar to ATPase recovery. However, the time courses could be better fitted by a double exponential function with comparable intensities than by a single exponential function (Fig. 3C, red line and green line, respectively). This suggests the presence of multiple pathways or intermediates toward low FRET state. Two (fast and slow) rate constants obtained from the double exponential function increased in a parallel manner as ATP concentrations increased. The fast rate constants showed a good match with the rate constants of recovery of ATPase activity at all ATP concentrations (Fig. 3D, triangles and circles, respectively). However, the lower one was severalfold smaller (Fig. 3D, inverted triangles).
      Temperature Dependence of Conformational Change—Physiological temperature of thermophilic Bacillus PS3 is about 65 °C, and F1 from this strain exhibits highest ATPase activity around this temperature (
      • Yoshida M.
      • Sone N.
      • Hirata H.
      • Kagawa Y.
      ). Next, temperature dependence of the rate of the conformational change of the ϵ subunit was investigated. An increase and decrease in donor fluorescence were induced by addition of ATP (1 mm) and by ATP depletion, respectively. Apparently, changes in both directions were strongly dependent on temperature. To reach a half-maximum fluorescence intensity, it took 95 s (increase) and 1300 s (decrease) at 30 °C, and 7.4 s (increase) and 130 s (decrease) at 45 °C.
      Time courses for both increase and decrease were better fitted by the double exponential function at all temperatures. Values of four rate constants (fast and slow components for both directions) increased as temperature increased in almost parallel way and the Arrhenius plots were linear in the temperature range from 30 to 45 °C (Fig. 4, C and D). The Arrhenius activation energies ranged from 88 to 126 kJ/mol, and high Arrhenius activation energy is consistent with large conformational change of the ϵ subunit. Although measurement at 65 °C was difficult, since the ATP regenerating system and hexokinase were rapidly inactivated, if we assumed that the linearity of the Arrhrenius plot was still retained at 65 °C, the extrapolated rate constants (faster one out of two rate constants) for the transitions reached to 2.7 s-1 (extended-to-retracted) and 0.18 s-1 (retracted-to-extended). These rates are much slower than that of ATPase turnover (>1000 s-1).
      Affinity of Nucleotides to the Isolated ϵ Subunit—Our previous study using gel chromatography showed that the isolated ϵ subunit binds ATP with relatively high affinity at 25 °C (the dissociation constant was expected to be lower than 10 μm) (
      • Kato-Yamada Y.
      • Yoshida M.
      ). However, quantitative estimation of the dissociation constant by gel chromatography was difficult. In this study, we applied fluorescence technique to detect nucleotide binding to the isolated ϵ subunit. When Cy3 was conjugated with ϵ(Q107C), a mutant that has a cysteine residue at the loop between two α-helices of the ϵ subunit, addition of a high concentration of nucleotide caused an increase in the fluorescence intensity of Cy3 several folds, presumably due to the environmental change around the fluorophore induced by nucleotide binding (Fig. 5A). If the change in the fluorescence was plotted against concentration of nucleotide, the plots were well fitted by the function assuming the simple binding reaction (Fig. 5B). The dissociation constants of ATP and ADP to the isolated ϵ subunit at 36 °C, estimated by the fitting, were 1.4 and 130 μm, respectively. The affinity of ATP was about 100 times higher than that of ADP. Affinity of GTP was much lower than that of ATP, and the apparent dissociation constant at 36 °C was 1.8 mm. Note that the value of dissociation constant for GTP is a lower limit, since contaminated ATP in GTP (<0.1% in our sample) could bind to the ϵ subunit and increase the Cy3 fluorescence.
      Temperature dependence of the affinity between ATP and the isolated ϵ subunit was also investigated. The dissociation constant between ATP and the isolated ϵ subunit increased greatly as temperature increased (Fig. 5C), and the logarithmic plot of dissociation constant against reciprocal temperature showed linear relationship (Fig. 5D). The dissociation constant extrapolated to the physiological temperature (65 °C) was 0.67 mm.

      DISCUSSION

      Conformational Change of the ϵ Subunit in F1 Is a Slow Process—Although a large conformational change of the ϵ subunit in F1 (and F0F1-ATP synthase) has been suggested from previous structural and biochemical studies (
      • Uhlin U.
      • Cox G.B.
      • Guss J.M.
      ,
      • Wilkens S.
      • Capaldi R.A.
      ,
      • Gibbons C.
      • Montgomery M.G.
      • Leslie A.G.
      • Walker J.E.
      ,
      • Rodgers A.J.
      • Wilce M.C.
      ,
      • Hausrath A.C.
      • Capaldi R.A.
      • Matthews B.W.
      ,
      • Tsunoda S.P.
      • Rodgers A.J.
      • Aggeler R.
      • Wilce M.C.
      • Yoshida M.
      • Capaldi R.A.
      ,
      • Suzuki T.
      • Murakami T.
      • Iino R.
      • Suzuki J.
      • Ono S.
      • Shirakihara Y.
      • Yoshida M.
      ,
      • Kato-Yamada Y.
      • Yoshida M.
      • Hisabori T.
      ), FRET measurement for the first time enabled us to observe it in real time. FRET measurement showed that even in the presence of a saturating concentrations of ATP, the change from high FRET to low FRET takes >10 s (at 35 °C, Fig. 3D). Thus, the ATP-induced conformational change of the ϵ subunit in F1 is much slower than the catalytic turnover (
      • Yasuda R.
      • Noji H.
      • Yoshida M.
      • Kinosita Jr., K.
      • Itoh H.
      ), suggesting that it cannot be one of obligate steps in each cycle of the catalytic turnover but rather it exerts a switch-like or gear change function of the catalysis.
      Conformational Change of the ϵ Subunit in F1 Could Occur at the 80 ° Substep Position—As we reported previously, ATP binding to the β subunit induces an 80° rotation of the γ subunit (
      • Yasuda R.
      • Noji H.
      • Yoshida M.
      • Kinosita Jr., K.
      • Itoh H.
      ). Subsequently the hydrolysis of ATP and another catalytic event (probably the release of ADP or phosphate) occur in 2 ms at this 80° position (catalytic dwell) (
      • Shimabukuro K.
      • Yasuda R.
      • Muneyuki E.
      • Hara K.Y.
      • Kinosita Jr., K.
      • Yoshida M.
      ), and the next 40° rotation follows to complete a 120° rotation, a unit rotation driven by single turnover of ATP hydrolysis. Also it was shown that F1 lapses into ADP-Mg inhibited state at the 80° position (
      • Hirono-Hara Y.
      • Noji H.
      • Nishiura M.
      • Muneyuki E.
      • Hara K.Y.
      • Yasuda R.
      • Kinosita Jr., K.
      • Yoshida M.
      ). We observed that not only ATP, but also AMPPNP, ADP + NaN3 and ADP + AlF4- induced the transition from high FRET to low FRET (Fig. 2B). These nucleotide conditions are thought to mimic the ATP-bound state (
      • Abrahams J.P.
      • Leslie A.G.
      • Lutter R.
      • Walker J.E.
      ,
      • Yasuda R.
      • Masaike T.
      • Adachi K.
      • Noji H.
      • Itoh H.
      • Kinosita Jr., K.
      ), the ADP-inhibited state (
      • Muneyuki E.
      • Makino M.
      • Kamata H.
      • Kagawa Y.
      • Yoshida M.
      • Hirata H.
      ), and the catalytic intermediate (
      • Braig K.
      • Menz R.I.
      • Montgomery M.G.
      • Leslie A.G.
      • Walker J.E.
      ), respectively, and stabilize the 80° position. Therefore, it is likely that transition of α-helices of the ϵ subunit from the extended form to the retracted form can take place when the γ subunit in F1 dwells at the 80° position.
      Relation between Recovery from Inhibition and Conformational Change of the ϵ Subunit—Recovery of ATPase from the inhibition by the ϵ subunit and FRET change induced by ATP addition occurred in a similar time scale, but their time courses did not agree each other. The real reason of this disagreement is not known. One of possible reasons might be ADP-Mg inhibition. In an attempt to avoid ADP-Mg inhibition, we supplemented a detergent, lauryldodecylamine oxide, to the solution of ATPase assay and FRET measurement and repeated the experiments of Figs. 3 and 4 (supplemental Figs. S1 and S2). Lauryldodecylamine oxide has been thought to help F1 not to lapse into ADP-Mg inhibition (
      • Paik S.R.
      • Jault J.M.
      • Allison W.S.
      ). The FRET change became faster in the presence of lauryldodecylamine oxide, while the time course of recovery of ATPase was almost unchanged
      ATP binding to the isolated ϵ subunit was not affected by lauryldodecylamine oxide (Kd = 1.4 μm at 35 °C).
      . However, the kinetics of ATPase recovery and FRET change still showed disagreement. We suspect that our F1 preparation could be heterogeneous, and some of the molecules that do not contain the ϵ subunit would be activated from the beginning. We expect that FRET observation at the single molecule level will reveal any heterogeneous population of F1 preparation.
      ATP Binding to the ϵ Subunit May Stabilize the Folded-hairpin Form—The isolated ϵ subunit of F1 from thermophilic Bacillus PS3 binds ATP (
      • Kato-Yamada Y.
      • Yoshida M.
      ), and our recent crystal structure of the ϵ-ATP complex has revealed that ATP binds to the ϵ subunit in a retracted conformation with folded-hairpin helices.
      N. Kajiwara, H. Akutsu, Y. Kato-Yamada, and M. Yoshida, unpublished data.
      Therefore, we assume that ATP binding to the ϵ subunit may stabilize the folded-hairpin form of the ϵ subunit in F1 to shift the equilibrium from the extended form. In the absence of ATP, the ϵ subunit adopts the extended form in most of time and transient conformational change induced by thermal fluctuation might be rare. A likely scenario is that ATP binding to the β subunits enhances the chance more often for the ϵ subunit with the extended form to make conformational transition to the folded-hairpin form to which another ATP binds subsequently, and the folded-hairpin form is stabilized. Non-catalytic nucleotide binding sites on the α subunits are not involved in this change because their deficient mutant F1(ΔNC) (
      • Matsui T.
      • Muneyuki E.
      • Honda M.
      • Allison W.S.
      • Dou C.
      • Yoshida M.
      ) also exhibited conformational change by ATP (data not shown) (
      • Kato-Yamada Y.
      • Yoshida M.
      • Hisabori T.
      ). A model of conformational dynamics of the ϵ subunit at the physiological temperature of thermophilic Bacillus PS3 (65 °C) is shown in Fig. 6. It should be noted that relatively weak ATP binding affinity of the ϵ subunit at the physiological temperature (Kd = 0.67 mm at 65 °C) is suitable to sense the cellular ATP concentration that is in submillimolar to millimolar range. This weak affinity can explain why the ATP binding to the ϵ subunit was detected only for thermophilic Bacillus PS3 but not for other species such as Escherichia coli (
      • Kato-Yamada Y.
      • Yoshida M.
      ). If the ϵ subunit from mesophilic organisms living below 40 °C would have similar weak affinity to ATP, the binding would not be detected by gel chromatography at room temperature.
      Figure thumbnail gr6
      FIGURE 6A model of conformational dynamics of the ϵ in F1. In this model, the rate constants (fast component) and the dissociation constant at the physiological temperature (65 °C) are shown. It is assumed that ATP binding to the catalytic β subunits, not to the ϵ subunits, induces conformational change to the retracted form. Lowering ATP concentration induces the reverse transition to the extended from. The dissociation constant between the ϵ and ATP is 0.67 mm, and direct binding of ATP to the ϵ subunit may stabilize the retracted form. The time scales of conformational transition (1/kretract and 1/kextend, >100 ms) are much longer than that of ATP hydrolysis by F1 (∼1 ms at 25 °C).
      The ϵ Subunit, an Inhibitory or a Coupling Factor?—Even at 0.1 mm ATP, almost all ϵ subunit molecules adopt the retracted form in a couple of min after initiation of ATP hydrolysis (Fig. 3B), and an inhibitory effect of the ϵ subunit was observed only for a short period (Fig. 3A). However, the situation may be different for F0F1-ATP synthase (F0F1) working in vivo. In our previous cross-linking experiment, a significant population of the ϵ subunit in F0F1 adopted the fully extended form even in the presence of relatively high concentrations of ATP (for example, ∼40% of the ϵ subunit was in the fully extended form at 1 mm ATP) (
      • Suzuki T.
      • Murakami T.
      • Iino R.
      • Suzuki J.
      • Ono S.
      • Shirakihara Y.
      • Yoshida M.
      ). The population was further increased by the proton motive force across the membrane. Therefore, compared with F1, the equilibrium of the conformation of the ϵ subunit in F0F1 must be more shifted to the fully extended form in vivo, and the ϵ subunit likely inhibits ATP hydrolysis more effectively. Furthermore, it was shown recently that reconstitution of the ϵ subunit into the α3β3γ subcomplex greatly improved the efficiency of ATP synthesis when F1 was rotated in the ATP synthesis direction with magnetic tweezers (
      • Rondelez Y.
      • Tresset G.
      • Nakashima T.
      • Kato-Yamada Y.
      • Fujita H.
      • Takeuchi S.
      • Noji H.
      ). This result indicates that the ϵ subunit acts not only as an endogenous inhibitor of ATP hydrolysis but also as a coupling factor for the ATP synthesis, as suggested before (
      • Cipriano D.J.
      • Bi Y.
      • Dunn S.D.
      ).
      Although the importance of the ϵ subunit is evident, it is totally unknown how the proton motive force affects the conformation of the ϵ subunit and how the ϵ subunit improves the efficiency of ATP synthesis. Rotational direction of the central stalk consisting of the γϵc10 complex, i.e. the direction of force applied to the ϵ subunit, may affect its conformation and function in non-equilibrium condition. Recent single molecule FRET measurements indicated that there was no conformational change in the N-terminal β-sandwitch part of the ϵ subunit during ATP hydrolysis and synthesis reaction catalyzed by F0F1 from E. coli (
      • Zimmermann B.
      • Diez M.
      • Zarrabi N.
      • Graber P.
      • Borsch M.
      ). The difference in the conformational states and dynamics of the C-terminal α-helices of the ϵ subunit during ATP hydrolysis and synthesis also would be directly probed by the single molecule FRET measurement during the forced rotation of F0F1.

      Acknowledgments

      We thank Drs. T. Masaike, K. Shimabukuro, N. Mitome, H. Ueno, and H. Noji for helpful discussions.

      References

        • Boyer P.D.
        Annu. Rev. Biochem. 1997; 66: 717-749
        • Yoshida M.
        • Muneyuki E.
        • Hisabori T.
        Nat. Rev. Mol. Cell. Biol. 2001; 2: 669-677
        • Uhlin U.
        • Cox G.B.
        • Guss J.M.
        Structure (Camb.). 1997; 5: 1219-1230
        • Wilkens S.
        • Capaldi R.A.
        J. Biol. Chem. 1998; 273: 26645-26651
        • Gibbons C.
        • Montgomery M.G.
        • Leslie A.G.
        • Walker J.E.
        Nat. Struct. Biol. 2000; 7: 1055-1061
        • Rodgers A.J.
        • Wilce M.C.
        Nat. Struct. Biol. 2000; 7: 1051-1054
        • Hausrath A.C.
        • Capaldi R.A.
        • Matthews B.W.
        J. Biol. Chem. 2001; 276: 47227-47232
        • Tsunoda S.P.
        • Rodgers A.J.
        • Aggeler R.
        • Wilce M.C.
        • Yoshida M.
        • Capaldi R.A.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6560-6564
        • Suzuki T.
        • Murakami T.
        • Iino R.
        • Suzuki J.
        • Ono S.
        • Shirakihara Y.
        • Yoshida M.
        J. Biol. Chem. 2003; 278: 46840-46846
        • Stryer L.
        Annu. Rev. Biochem. 1978; 47: 819-846
        • Shimabukuro K.
        • Yasuda R.
        • Muneyuki E.
        • Hara K.Y.
        • Kinosita Jr., K.
        • Yoshida M.
        Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14731-14736
        • Kato-Yamada Y.
        • Yoshida M.
        J. Biol. Chem. 2003; 278: 36013-36016
        • Abrahams J.P.
        • Leslie A.G.
        • Lutter R.
        • Walker J.E.
        Nature. 1994; 370: 621-628
        • Muneyuki E.
        • Makino M.
        • Kamata H.
        • Kagawa Y.
        • Yoshida M.
        • Hirata H.
        Biochim. Biophys. Acta. 1993; 1144: 62-68
        • Braig K.
        • Menz R.I.
        • Montgomery M.G.
        • Leslie A.G.
        • Walker J.E.
        Struct. Fold. Des. 2000; 8: 567-573
        • Noji H.
        • Bald D.
        • Yasuda R.
        • Itoh H.
        • Yoshida M.
        • Kinosita Jr., K.
        J. Biol. Chem. 2001; 276: 25480-25486
        • Kato Y.
        • Matsui T.
        • Tanaka N.
        • Muneyuki E.
        • Hisabori T.
        • Yoshida M.
        J. Biol. Chem. 1997; 272: 24906-24912
        • Yoshida M.
        • Sone N.
        • Hirata H.
        • Kagawa Y.
        J. Biol. Chem. 1975; 250: 7910-7916
        • Kato-Yamada Y.
        • Yoshida M.
        • Hisabori T.
        J. Biol. Chem. 2000; 275: 35746-35750
        • Yasuda R.
        • Noji H.
        • Yoshida M.
        • Kinosita Jr., K.
        • Itoh H.
        Nature. 2001; 410: 898-904
        • Hirono-Hara Y.
        • Noji H.
        • Nishiura M.
        • Muneyuki E.
        • Hara K.Y.
        • Yasuda R.
        • Kinosita Jr., K.
        • Yoshida M.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13649-13654
        • Yasuda R.
        • Masaike T.
        • Adachi K.
        • Noji H.
        • Itoh H.
        • Kinosita Jr., K.
        Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9314-9318
        • Paik S.R.
        • Jault J.M.
        • Allison W.S.
        Biochemistry. 1994; 33: 126-133
        • Matsui T.
        • Muneyuki E.
        • Honda M.
        • Allison W.S.
        • Dou C.
        • Yoshida M.
        J. Biol. Chem. 1997; 272: 8215-8221
        • Rondelez Y.
        • Tresset G.
        • Nakashima T.
        • Kato-Yamada Y.
        • Fujita H.
        • Takeuchi S.
        • Noji H.
        Nature. 2005; 433: 773-777
        • Cipriano D.J.
        • Bi Y.
        • Dunn S.D.
        J. Biol. Chem. 2002; 277: 16782-16790
        • Zimmermann B.
        • Diez M.
        • Zarrabi N.
        • Graber P.
        • Borsch M.
        EMBO J. 2005; 24: 2053-2063