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Activation and Stiffness of the Inhibited States of F1-ATPase Probed by Single-molecule Manipulation

Open AccessPublished:February 12, 2010DOI:https://doi.org/10.1074/jbc.M109.099143
      F1-ATPase (F1), a soluble portion of FoF1-ATP synthase (FoF1), is an ATP-driven motor in which γϵ subunits rotate in the α3β3 cylinder. Activity of F1 and FoF1 from Bacillus PS3 is attenuated by the ϵ subunit in an inhibitory extended form. In this study we observed ATP-dependent transition of ϵ in single F1 molecules from extended form to hairpin form by fluorescence resonance energy transfer. The results justify the previous bulk experiments and ensure that fraction of F1 with hairpin ϵ directly determines the fraction of active F1 at any ATP concentration. Next, mechanical activation and stiffness of ϵ-inhibited F1 were examined by the forced rotation of magnetic beads attached to γ. Compared with ADP inhibition, which is another manner of inhibition, rotation by a larger angle was required for the activation from ϵ inhibition when the beads were forced to rotate to ATP hydrolysis direction, and more torque was required to reach the same rotation angle when beads were forced to rotate to ATP synthesis direction. The results imply that if FoF1 is resting in the ϵ-inhibited state, Fo motor must transmit to γ a torque larger than expected from thermodynamic equilibrium to initiate ATP synthesis.

      Introduction

      FoF1-ATP synthase (FoF1) is a ubiquitous enzyme located in bacterial plasma membranes, mitochondrial inner membranes, and chloroplast thylakoid membranes. FoF1 consists of two major portions, water-soluble F1 and membrane-embedded Fo. In the simplest version of bacterial FoF1 such as FoF1 from thermophilic Bacillus PS3 and Escherichia coli, subunit composition of F1 and Fo is α3β3γδϵ and ab2c10, respectively. Downward proton flow across membrane through Fo along proton motive force (pmf)
      The abbreviations used are: pmf
      proton motive force
      FRET
      fluorescence resonance energy transfer
      MCV
      minimum clamping voltage.
      drives the rotation of the rotor ring of oligomer c subunits (c-ring) in Fo that induces rotation of the rotor shaft of F1 consisting of the γ and ϵ subunits in the surrounding α3β3 cylinder. This rotation causes cyclic conformational changes in the catalytic sites in β subunits that result in ATP synthesis (
      • Boyer P.D.
      ,
      • Yoshida M.
      • Muneyuki E.
      • Hisabori T.
      ,
      • Diez M.
      • Zimmermann B.
      • Börsch M.
      • König M.
      • Schweinberger E.
      • Steigmiller S.
      • Reuter R.
      • Felekyan S.
      • Kudryavtsev V.
      • Seidel C.A.
      • Gräber P.
      ,
      • Nakamoto R.K.
      • Baylis Scanlon J.A.
      • Al-Shawi M.K.
      ,
      • von Ballmoos C.
      • Wiedenmann A.
      • Dimroth P.
      ). FoF1 can catalyze a back reaction, ATP hydrolysis-driven proton pumping, when ATP hydrolysis is thermodynamically more favorable than pmf-driven proton flow. The isolated F1 has ATPase activity, often called F1-ATPase, and is itself an ATP-driven rotary motor (
      • Duncan T.M.
      • Bulygin V.V.
      • Zhou Y.
      • Hutcheon M.L.
      • Cross R.L.
      ,
      • Noji H.
      • Yasuda R.
      • Yoshida M.
      • Kinosita Jr., K.
      ,
      • Spetzler D.
      • York J.
      • Daniel D.
      • Fromme R.
      • Lowry D.
      • Frasch W.
      ). Starting from the orientation angle of the γ subunit at 0° (ATP-waiting dwell), ATP binding induces the 80° substep rotation of γ, the ATP previously bound is hydrolyzed (catalytic dwell), and Pi-release induces the 40° substep rotation (
      • Yasuda R.
      • Noji H.
      • Yoshida M.
      • Kinosita Jr., K.
      • Itoh H.
      ,
      • Ariga T.
      • Muneyuki E.
      • Yoshida M.
      ,
      • Adachi K.
      • Oiwa K.
      • Nishizaka T.
      • Furuike S.
      • Noji H.
      • Itoh H.
      • Yoshida M.
      • Kinosita Jr., K.
      ). Duration of ATP-waiting dwell is inversely proportional to the concentration of ATP ([ATP]), but that of the catalytic dwell is independent from ATP, always a few milliseconds.
      ATPase activity of F1 as well as FoF1 is attenuated by several mechanisms. In bacteria, two mechanisms are common; they are inhibition by MgADP (ADP inhibition) and inhibition by ϵ subunit (ϵ inhibition). ADP inhibition is caused by the persistent occupation of a high affinity catalytic site by MgADP (without Pi) (
      • Dunham K.R.
      • Selman B.R.
      ,
      • Carmeli C.
      • Lifshitz Y.
      ,
      • Drobinskaya I.Y.
      • Kozlov I.A.
      • Murataliev M.B.
      • Vulfson E.N.
      ,
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      • Suzuki T.
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      ,
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      ,
      • Jault J.M.
      • Allison W.S.
      ,
      • Bandyopadhyay S.
      • Muneyuki E.
      • Allison W.S.
      ). Using Bacillus PS3 F1, Hirono-Hara et al. revealed that ADP inhibition accompanied the stall of rotary catalysis at the catalytic dwell angle (
      • Hirono-Hara Y.
      • Noji H.
      • Nishiura M.
      • Muneyuki E.
      • Hara K.Y.
      • Yasuda R.
      • Kinosita Jr., K.
      • Yoshida M.
      ) and that activation required rotation of γ by ∼40° in ATP hydrolysis direction by mechanical manipulation (
      • Hirono-Hara Y.
      • Ishizuka K.
      • Kinosita Jr., K.
      • Yoshida M.
      • Noji H.
      ) or by thermal fluctuation without manipulation. In the case of ATP synthesis, high pmf can expel the inhibitory ADP from FoF1, and once activated, turnover of ATP synthesis is not interrupted by ADP inhibition (for details, see review Ref
      • Feniouk B.A.
      • Yoshida M.
      and references therein). The ϵ subunit is part of a rotor (
      • Kato-Yamada Y.
      • Noji H.
      • Yasuda R.
      • Kinosita Jr., K.
      • Yoshida M.
      ,
      • Bulygin V.V.
      • Duncan T.M.
      • Cross R.L.
      ) and has been known as an intrinsic inhibitor of ATPase activity (
      • Weber J.
      • Dunn S.D.
      • Senior A.E.
      ,
      • Kato Y.
      • Matsui T.
      • Tanaka N.
      • Muneyuki E.
      • Hisabori T.
      • Yoshida M.
      ) as well as a subunit that improves coupling efficiency (
      • Rondelez Y.
      • Tresset G.
      • Nakashima T.
      • Kato-Yamada Y.
      • Fujita H.
      • Takeuchi S.
      • Noji H.
      ,
      • Cipriano D.J.
      • Dunn S.D.
      ). The ϵ subunit consists of two domains (
      • Uhlin U.
      • Cox G.B.
      • Guss J.M.
      ,
      • Wilkens S.
      • Capaldi R.A.
      ); that is, an N-terminal β-sandwich domain, by which ϵ binds to globular part of γ and c-ring, and a C-terminal domain with two α-helices, which undergoes conformational transition between hairpin and extended forms (
      • Rodgers A.J.
      • Wilce M.C.
      ,
      • Kato-Yamada Y.
      • Yoshida M.
      • Hisabori T.
      ,
      • Ganti S.
      • Vik S.B.
      ,
      • Bulygin V.V.
      • Duncan T.M.
      • Cross R.L.
      ,
      • Suzuki T.
      • Murakami T.
      • Iino R.
      • Suzuki J.
      • Ono S.
      • Shirakihara Y.
      • Yoshida M.
      ). ATPase activity is inhibited only when the two α-helices are extended. In ϵ inhibition, rotation is slowed down (
      • Nakanishi-Matsui M.
      • Kashiwagi S.
      • Hosokawa H.
      • Cipriano D.J.
      • Dunn S.D.
      • Wada Y.
      • Futai M.
      ) or paused at the position of the catalytic dwell, the same position as observed for ADP inhibition (
      • Konno H.
      • Murakami-Fuse T.
      • Fujii F.
      • Koyama F.
      • Ueoka-Nakanishi H.
      • Pack C.G.
      • Kinjo M.
      • Hisabori T.
      ,
      • Tsumuraya M.
      • Furuike S.
      • Adachi K.
      • Kinosita Jr., K.
      • Yoshida M.
      ), and it has been proposed that ϵ inhibition involves strengthened ADP inhibition (
      • Feniouk B.A.
      • Suzuki T.
      • Yoshida M.
      ,
      • Tsumuraya M.
      • Furuike S.
      • Adachi K.
      • Kinosita Jr., K.
      • Yoshida M.
      ). The manner of ϵ inhibition varies among species. In the case of Bacillus PS3 F1, the inhibition is observed only at low [ATP] and ATP can bind to and stabilize the non-inhibitory hairpin form of ϵ. ATP synthesis reaction appears to be affected by C-terminal domain of ϵ as a mutant FoF1ΔC) that lacked this domain had a higher rate of ATP synthesis (
      • Masaike T.
      • Suzuki T.
      • Tsunoda S.P.
      • Konno H.
      • Yoshida M.
      ,
      • Iino R.
      • Hasegawa R.
      • Tabata K.V.
      • Noji H.
      ).
      In this study, a population of hairpin ϵ in Bacillus PS3 F1 immobilized on the glass surface was directly estimated at various [ATP] by single-pair fluorescence resonance energy transfer (FRET). The results confirmed the previous bulk FRET experiments and correspondence of the fraction of F1 containing the hairpin ϵ to the fraction of active F1 at any [ATP]. Next, magnetic beads attached to γ of ϵ-inhibited and ADP-inhibited F1 were forced to rotate by a external magnetic field, and mechanical activation and stiffness of the inhibited forms were examined. The results indicate that the ϵ-inhibited F1 is more resistant against mechanical perturbation than ADP-inhibited F1, and more torque is required for activation.

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