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Single-molecule Study on the Temperature-sensitive Reaction of F1-ATPase with a Hybrid F1 Carrying a Single β(E190D)*

  • Sawako Enoki
    Affiliations
    From the Institute of Scientific and Industrial Research, Osaka University, 567-0047 Osaka, Japan
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  • Rikiya Watanabe
    Affiliations
    From the Institute of Scientific and Industrial Research, Osaka University, 567-0047 Osaka, Japan
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  • Ryota Iino
    Affiliations
    From the Institute of Scientific and Industrial Research, Osaka University, 567-0047 Osaka, Japan
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  • Hiroyuki Noji
    Correspondence
    To whom correspondence should be addressed. Tel.: 81-6-6879-8481; Fax: 81-6-6875-5724; E-mail: [email protected]
    Affiliations
    From the Institute of Scientific and Industrial Research, Osaka University, 567-0047 Osaka, Japan
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  • Author Footnotes
    * This work was supported by Grants-in-aid 18074005 and 18201025 for Scientific Research (to H. N.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by a grant from the Post-Silicon Materials and Devices Research Alliance, Institute of Scientific and Industrial Research at Osaka University (to H. N.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.
Open AccessPublished:June 26, 2009DOI:https://doi.org/10.1074/jbc.M109.026401
      F1-ATPase is a rotary molecular motor in which the γ-subunit rotates against the α3β3 cylinder. The unitary γ-rotation is a 120° step comprising 80 and 40° substeps, each of these initiated by ATP binding and ADP release and by ATP hydrolysis and inorganic phosphate release, respectively. In our previous study on γ-rotation at low temperatures, a highly temperature-sensitive (TS) reaction step of F1-ATPase from thermophilic Bacillus PS3 was found below 9 °C as an intervening pause before the 80° substep at the same angle for ATP binding and ADP release. However, it remains unclear as to which reaction step the TS reaction corresponds. In this study, we found that the mutant F1(βE190D) from thermophilic Bacillus PS3 showed a clear pause of the TS reaction below 18 °C. In an attempt to identify the catalytic state of the TS reaction, the rotation of the hybrid F1, carrying a single copy of βE190D, was observed at 18 °C. The hybrid F1 showed a pause of the TS reaction at the same angle as for the ATP binding of the incorporated βE190D, although kinetic analysis revealed that the TS reaction is not the ATP binding step. These findings suggest that the TS reaction is a structural rearrangement of β before or after ATP binding.
      F1-ATPase (F1)
      The abbreviations used are: F1
      F1-ATPase
      ATPγS
      adenosine 5′-O-(thiotriphosphate)
      TS
      temperature-sensitive.
      2The abbreviations used are: F1
      F1-ATPase
      ATPγS
      adenosine 5′-O-(thiotriphosphate)
      TS
      temperature-sensitive.
      is an ATP-driven rotary motor protein. The subunit composition of the bacterial F1-ATPase is α3β3γδϵ, and the minimum complex of F1-ATPase as a rotary motor is α3β3γ subcomplex. This motor protein forms the FoF1-ATP synthase complex by binding to another rotary motor, namely, Fo, which is driven by the proton flux resulting from the proton motive force across the membranes (
      • Boyer P.D.
      ,
      • Cross R.L.
      ,
      • Senior A.E.
      • Nadanaciva S.
      • Weber J.
      ,
      • Yoshida M.
      • Muneyuki E.
      • Hisabori T.
      ). Under physiological conditions, where the proton motive force is sufficiently large, Fo forcibly rotates F1-ATPase in the reverse direction of F1-ATPase, leading the reverse reaction of ATP hydrolysis, i.e. ATP synthesis from ADP and inorganic phosphate (Pi). When the proton motive force diminishes or F1 is isolated from Fo, F1-ATPase hydrolyzes ATP to rotate the γ-subunit against the α3β3 stator ring in the counterclockwise direction as viewed from the Fo side (
      • Noji H.
      • Yasuda R.
      • Yoshida M.
      • Kinosita Jr., K.
      ). The catalytic sites are located at the interface of the α- and β-subunits, predominantly on the β-subunit (
      • Abrahams J.P.
      • Leslie A.G.
      • Lutter R.
      • Walker J.E.
      ). Each β-subunit carries out a single turnover of ATP hydrolysis during the γ-rotation of 360° following the common catalytic reaction pathway, whereas they are 120° different in the catalytic phase. In this manner, the three β-subunits undergo different reaction steps of ATP hydrolysis upon each rotational step. The rotary motion of the γ-subunit has been demonstrated by biochemical (
      • Duncan T.M.
      • Bulygin V.V.
      • Zhou Y.
      • Hutcheon M.L.
      • Cross R.L.
      ) and spectroscopic methods (
      • Sabbert D.
      • Engelbrecht S.
      • Junge W.
      ) and directly proved in single-molecule observation studies (
      • Noji H.
      • Yasuda R.
      • Yoshida M.
      • Kinosita Jr., K.
      ).
      Since the establishment of the single-molecule rotation assay, the chemomechanical coupling scheme of F1 has been studied extensively by resolving the rotation into discrete steps. The stepping rotation was first observed under an ATP-limiting condition where F1 makes discrete 120° steps upon ATP binding (
      • Yasuda R.
      • Noji H.
      • Kinosita Jr., K.
      • Yoshida M.
      ). Then, high speed imaging of the rotation with a small probe of low friction was performed, which revealed that the 120° step comprises 80 and 40° substeps, each initiated by ATP binding, and two unknown consecutive reactions, respectively (
      • Yasuda R.
      • Noji H.
      • Yoshida M.
      • Kinosita Jr., K.
      • Itoh H.
      ). This finding necessitated the identification of the two reactions that trigger the 40° substep. Hence, the rotation assay was performed using a mutant, namely F1(βE190D), and a slowly hydrolyzed ATP analog, namely ATPγS (
      • Shimabukuro K.
      • Yasuda R.
      • Muneyuki E.
      • Hara K.Y.
      • Kinosita Jr., K.
      • Yoshida M.
      ). Glutamate 190 of the β-subunit of F1, derived from thermophilic Bacillus PS3 and the corresponding glutamates from other F1-ATPases (Glu-181 of F1 from Escherichia coli and Glu-188 of F1 from bovine mitochondria), has been identified as one of the most critical catalytic residues for ATP hydrolysis (
      • Abrahams J.P.
      • Leslie A.G.
      • Lutter R.
      • Walker J.E.
      ,
      • Amano T.
      • Tozawa K.
      • Yoshida M.
      • Murakami H.
      ,
      • Ohtsubo M.
      • Yoshida M.
      • Ohta S.
      • Kagawa Y.
      • Yohda M.
      • Date T.
      ,
      • Park M.Y.
      • Omote H.
      • Maeda M.
      • Futai M.
      ,
      • Senior A.E.
      • al-Shawi M.K.
      ). When this glutamate was substituted with aspartic acid, which has a shorter side chain than that of glutamate, the ATP cleavage step of F1 was drastically slowed. In the rotation assay, this mutant showed a distinct long pause before the 40° substep. ATPγS also caused a long pause before the 40° substep. These observations established that the 40° substep is initiated by hydrolysis. Accordingly, the pause angles before the 80 and 40° substeps are referred to as to the binding angle and the catalytic angle, respectively. Then, the rotation assay was performed in the presence of a high amount of Pi in the solution. It was shown that Pi rebinding caused the long pause at the catalytic angle, suggesting that Pi is released before the 40° substep (
      • Adachi K.
      • Oiwa K.
      • Nishizaka T.
      • Furuike S.
      • Noji H.
      • Itoh H.
      • Yoshida M.
      • Kinosita Jr., K.
      ).
      However, the reaction scheme of F1 cannot be established by simply assigning each reaction step to either the binding angle or the catalytic angle, because each reaction step must be assigned to one of the three binding or catalytic angles when considering the 360° cyclic reaction scheme of each β-subunit. Direct information about the timing of ADP release was obtained by simultaneous imaging of fluorescently labeled nucleotides and γ rotation, which showed that each β retains ADP until the γ rotates 240° after binding of the nucleotide as ATP and releases ADP between 240 and 320° (
      • Adachi K.
      • Oiwa K.
      • Nishizaka T.
      • Furuike S.
      • Noji H.
      • Itoh H.
      • Yoshida M.
      • Kinosita Jr., K.
      ,
      • Nishizaka T.
      • Oiwa K.
      • Noji H.
      • Kimura S.
      • Muneyuki E.
      • Yoshida M.
      • Kinosita Jr., K.
      ). Another powerful approach is the use of a hybrid F1 carrying a mutant β that causes a characteristic pause during the rotation. In a previous study, the hybrid F1 carrying a single copy of β(E190D), α3β2β(E190D)γ, showed a distinct pause caused by the slow hydrolysis of β(E190D) at +200° from the ATP binding angle of the mutant β (
      • Ariga T.
      • Muneyuki E.
      • Yoshida M.
      ). From this observation, it was confirmed that each β executes the chemical cleavage of the bound ATP at +200° from the angle where the ATP binds to β. The asymmetric feature of the pause of the hybrid F1 was also utilized in other experiments as a marker in the rotational trajectory to correlate the rotational angle and the conformational state of β (
      • Masaike T.
      • Koyama-Horibe F.
      • Oiwa K.
      • Yoshida M.
      • Nishizaka T.
      ) or to determine the state of F1 in the crystal structures as the pausing state at catalytic angle (
      • Okuno D.
      • Fujisawa R.
      • Iino R.
      • Hirono-Hara Y.
      • Imamura H.
      • Noji H.
      ).
      Recently, we have found a new reaction intermediate of F1 rotation as a clear intervening pause before the 80° substep in the rotation assay below 9 °C (
      • Watanabe R.
      • Iino R.
      • Shimabukuro K.
      • Yoshida M.
      • Noji H.
      ). Furuike et al. (
      • Furuike S.
      • Adachi K.
      • Sakaki N.
      • Shimo-Kon R.
      • Itoh H.
      • Muneyuki E.
      • Yoshida M.
      • Kinosita Jr., K.
      ) also observed the TS reaction in a high speed imaging experiment. The rate constant of this reaction was remarkably sensitive to temperature, giving a Q10 factor around 19. When ADP was added to solution, the pause before the 80° substep was prolonged, whereas the solution Pi caused a longer pause before the 40° substep (
      • Watanabe R.
      • Iino R.
      • Shimabukuro K.
      • Yoshida M.
      • Noji H.
      ). Although this result can be explained by assuming that the temperature-sensitive (TS) reaction is ADP release, it was not decisive for the identification of the TS reaction.
      In this study, we found that the mutant F1(βE190D) also exhibits the distinct pause of the TS reaction but at a higher temperature than for the wild-type F1, i.e. at 18 °C. This feature was advantageous in identifying the angle position of the TS reaction in the catalytic cycle for each β-subunit coupled with the 360° rotation. Taking advantage of the feature of the hybrid F1, we analyzed the rotational behavior of the hybrid F1 at 18 °C in order to assign the angle position of the TS reaction in the catalytic cycle of the 360° rotation, and we have shown that the TS reaction is not directly involved in the ADP release but in some conformational rearrangement before or after ATP binding step.

      DISCUSSION

      The TS reaction of F1(βE190D) was shown to have the same characteristics as the TS reaction of wild-type F1. (i) The reaction occurs at the ATP binding angle (FIGURE 2, FIGURE 3). (ii) The reaction strongly depends on temperature; the apparent activation energy of the reaction (78–86 kJ/mol) is as high as that of wild-type F1 (97–98 kJ/mol; B, Table 1). (iii) Finally, the reaction does not depend on the ATP concentration (Fig. 3). Thus, the temperature-sensitive reaction of F1(βE190D) is not the ATP binding step from solution. However, the rate constant of the TS reaction of F1(βE190D) was 150 times less than that of the wild type; therefore, it could be discriminated from the TS reaction of the wild type. Using this advantageous feature, the rotation of the hybrid F1, α3β2β(E190D)γ, which has a single copy of βE190D, was analyzed. It was clearly shown that the TS reaction occurs at the ATP binding angle, that is −200° (+160°) from the hydrolysis angle of the mutant β. This position corresponds to 0° in the 360° cyclic reaction scheme of the β-subunit. Based on this new finding, the reaction scheme including the TS reaction step was proposed (Fig. 5). Here, the TS reaction is assumed to be a conformational change of the β-subunit not coupled with a chemical reaction at the catalytic site because the β-subunit only carries out the ATP binding step at 0°, and there is strong evidence that the TS reaction is not the ATP binding step. There are at least two possible reaction schemes; the TS reaction could occur before or after ATP binding. The former model assumes that the TS reaction is the conformational change of the β-subunit to develop into the ATP binding site. The latter model assumes that the TS reaction is the conformational change of the β-subunit for the affinity change to bound ATP. The present study does not provide crucial data to discriminate between these models. One possible experimental approach for this task is the simultaneous imaging of the binding and release of a fluorescently labeled nucleotide and the γ-rotation at 18 °C.
      Figure thumbnail gr5
      FIGURE 5Proposed reaction scheme of F1 and the TS reaction. The β-subunit that binds ATP at 0° is shown as a dark green circle. The scheme at 0° where the TS reaction and ATP binding occur is shown in the upper panel (expanded). Two possible models are shown in parallel, the case of ATP binding following the TS reaction (upper model) and that in which the TS reaction follows ATP binding (lower model).
      Considering the exceptionally high temperature dependence of the TS reaction, it is likely that this reaction is a conformational rearrangement of the β-subunit, which accompanies a break of strong interaction. Therefore, it would seem that the TS reaction involves a global conformational rearrangement of the β-subunit. However, such a large conformational change during the 320–360° rotation has not been reported in a recent single-molecule work by Masaike et al. (
      • Masaike T.
      • Koyama-Horibe F.
      • Oiwa K.
      • Yoshida M.
      • Nishizaka T.
      ), who measured the angular displacement of the C-terminal domain of the β-subunit with advanced total internal reflection fluorescence microscopy. A possible explanation is that the TS reaction occurs after ATP binding and it was too fast for their time resolution (
      • Masaike T.
      • Koyama-Horibe F.
      • Oiwa K.
      • Yoshida M.
      • Nishizaka T.
      ) or that the TS reaction is accompanied by a conformational change in the horizontal plane of the rotational axis, which was not detectable with their method. Another idea of the TS reaction is a localized conformational rearrangement around the catalytic site. It seems to be reasonable considering that a single mutation introduced at glutamate 190 of the β-subunit causes the remarkable suppression in the rate constant of the TS reaction.
      In a previous paper, we suggested that the TS reaction is relevant to ADP release based on the observation that solution ADP prolongs the dwell time at the binding angle (
      • Watanabe R.
      • Iino R.
      • Shimabukuro K.
      • Yoshida M.
      • Noji H.
      ). However, the results in the present study indicate that the TS reaction is not directly related to the ADP release, which is thought to occur at +40° from the hydrolysis angle (+240° from the binding angle). The previous kinetic analysis was based on the assumption that solution ADP binds to the site for ADP release; however, it is also possible that solution ADP competes with ATP for the ATP binding site. This contention also explains the inhibitory effect of solution ADP on the TS reaction. In fact, although the pause of F1(βE190D) at the binding angle was prolonged by the addition of a large amount of ADP (20 mm) in solution, the same as the wild-type F1, the effect of ADP was largely suppressed by the addition of an excess amount of ATP (supplemental Fig. S3). These results indicate that for F1, solution ADP acts not as a product inhibitor but as a competitive inhibitor with ATP.
      It should be noted that there is a critical inconsistency between the present results and the previous report by Ariga et al. (
      • Ariga T.
      • Muneyuki E.
      • Yoshida M.
      ), who reported that the hybrid F1, α3β2β(E190D)γ rotates with one long pause at 200° caused by ATP hydrolysis and an additional short pause, as found in the present study. However, the angle position was assigned to be 320°, not 360°, although the time constant of their short pause was 20 ms (
      • Ariga T.
      • Muneyuki E.
      • Yoshida M.
      ), which is very close to the TS pause at 28 °C determined in the present experiment. Supplemental Fig. 2 shows the centroid plots for all hybrid F1 molecules for which the temperature change experiment was successfully carried out. In the data set, all molecules showed the short pause not at 320 but at 360°. Statistical analysis confirms this conclusion. The reason for this inconsistency is not clear. Although there are some small differences in the experimental procedures and materials, these do not seem to explain this critical inconsistency. Even if there is another short pause in addition to that of the TS reaction, the pause of the TS reaction should have been included in their data set.

      Acknowledgments

      We thank Dr. S. Nishikawa, Mr. K. Ikezaki, Dr. M. Sugawa, and Dr. T. Yanagida for technical help, Dr. K. Adachi for the custom image analysis program, Dr. T. Okamoto for critical discussion, and members of the Noji laboratory for help and advice.

      REFERENCES

        • Boyer P.D.
        Annu. Rev. Biochem. 1997; 66: 717-749
        • Cross R.L.
        Biochim. Biophys. Acta. 2000; 1458: 270-275
        • Senior A.E.
        • Nadanaciva S.
        • Weber J.
        Biochim. Biophys. Acta. 2002; 1553: 188-211
        • Yoshida M.
        • Muneyuki E.
        • Hisabori T.
        Nat. Rev. Mol. Cell Biol. 2001; 2: 669-677
        • Noji H.
        • Yasuda R.
        • Yoshida M.
        • Kinosita Jr., K.
        Nature. 1997; 386: 299-302
        • Abrahams J.P.
        • Leslie A.G.
        • Lutter R.
        • Walker J.E.
        Nature. 1994; 370: 621-628
        • Duncan T.M.
        • Bulygin V.V.
        • Zhou Y.
        • Hutcheon M.L.
        • Cross R.L.
        Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 10964-10968
        • Sabbert D.
        • Engelbrecht S.
        • Junge W.
        Nature. 1996; 381: 623-625
        • Yasuda R.
        • Noji H.
        • Kinosita Jr., K.
        • Yoshida M.
        Cell. 1998; 93: 1117-1124
        • Yasuda R.
        • Noji H.
        • Yoshida M.
        • Kinosita Jr., K.
        • Itoh H.
        Nature. 2001; 410: 898-904
        • 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
        • Amano T.
        • Tozawa K.
        • Yoshida M.
        • Murakami H.
        FEBS Lett. 1994; 348: 93-98
        • Ohtsubo M.
        • Yoshida M.
        • Ohta S.
        • Kagawa Y.
        • Yohda M.
        • Date T.
        Biochem. Biophys. Res. Commun. 1987; 146: 705-710
        • Park M.Y.
        • Omote H.
        • Maeda M.
        • Futai M.
        J. Biochem. 1994; 116: 1139-1145
        • Senior A.E.
        • al-Shawi M.K.
        J. Biol. Chem. 1992; 267: 21471-21478
        • Adachi K.
        • Oiwa K.
        • Nishizaka T.
        • Furuike S.
        • Noji H.
        • Itoh H.
        • Yoshida M.
        • Kinosita Jr., K.
        Cell. 2007; 130: 309-321
        • Nishizaka T.
        • Oiwa K.
        • Noji H.
        • Kimura S.
        • Muneyuki E.
        • Yoshida M.
        • Kinosita Jr., K.
        Nat. Struct. Mol. Biol. 2004; 11: 142-148
        • Ariga T.
        • Muneyuki E.
        • Yoshida M.
        Nat. Struct. Mol. Biol. 2007; 14: 841-846
        • Masaike T.
        • Koyama-Horibe F.
        • Oiwa K.
        • Yoshida M.
        • Nishizaka T.
        Nat. Struct. Mol. Biol. 2008; 15: 1326-1333
        • Okuno D.
        • Fujisawa R.
        • Iino R.
        • Hirono-Hara Y.
        • Imamura H.
        • Noji H.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 20722-20727
        • Watanabe R.
        • Iino R.
        • Shimabukuro K.
        • Yoshida M.
        • Noji H.
        EMBO Rep. 2008; 9: 84-90
        • Furuike S.
        • Adachi K.
        • Sakaki N.
        • Shimo-Kon R.
        • Itoh H.
        • Muneyuki E.
        • Yoshida M.
        • Kinosita Jr., K.
        Biophys. J. 2008; 95: 761-770
        • Hirono-Hara Y.
        • Ishizuka K.
        • Kinosita Jr., K.
        • Yoshida M.
        • Noji H.
        Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 4288-4293
        • Rondelez Y.
        • Tresset G.
        • Nakashima T.
        • Kato-Yamada Y.
        • Fujita H.
        • Takeuchi S.
        • Noji H.
        Nature. 2005; 433: 773-777