Mechanical modulation of catalytic power on F1-ATPase

Journal name:
Nature Chemical Biology
Volume:
8,
Pages:
86–92
Year published:
DOI:
doi:10.1038/nchembio.715
Received
Accepted
Published online

Abstract

The conformational fluctuation of enzymes has a crucial role in reaction acceleration. However, the contribution to catalysis enhancement of individual substates with conformations far from the average conformation remains unclear. We studied the catalytic power of the rotary molecular motor F1-ATPase from thermophilic Bacillus PS3 as it was stalled in transient conformations far from a stable pausing angle. The rate constants of ATP binding and hydrolysis were determined as functions of the rotary angle. Both rates exponentially increase with rotation, revealing the molecular basis of positive cooperativity among three catalytic sites: elementary reaction steps are accelerated via the mechanical rotation driven by other reactions on neighboring catalytic sites. The rate enhancement induced by ATP binding upon rotation was greater than that brought about by hydrolysis, suggesting that the ATP binding step contributes more to torque generation than does the hydrolysis step. Additionally, 9% of the ATP-driven rotary step was supported by thermal diffusion, suggesting that acceleration of the ATP docking process occurs via thermally agitated conformational fluctuations.

At a glance

Figures

  1. Experimental setup and procedure for manipulation of the F1 motor.
    Figure 1: Experimental setup and procedure for manipulation of the F1 motor.

    (a) Chemomechanical coupling scheme of F1. The circles and red arrows represent the catalytic states of β subunits and the angular positions of γ subunits. Each β subunit completes a single turnover of ATP hydrolysis with one turn of γ, whereas in the catalytic phase, three β subunits differ by 120°. The catalytic state of the top β subunit (cyan) is provided for clarification. ATP binding, hydrolysis, ADP release and Pi release occur at 0°, 200°, 240° and 320°, respectively. (b) Schematic image of the experimental setup (not to scale). (c) Experimental procedures for stalling experiment. When F1 paused the ATP binding dwell or hydrolysis dwell, the tweezers were turned on to stall F1 at the target angle and then were turned off to release the motor after the set period lapsed. A released motor shows rapid forward stepping (on) or a return to the original pause angle (off), behaviors indicating that the reaction under investigation is either complete or incomplete, respectively. (d) Examples of stalling experiment for ATP binding at 60 nM ATP. During a pause, F1 was stalled at +30° from the binding angle for 0.5 s and then released (Supplementary Movie 1, left side). After being released, F1 stepped to the next binding angle without moving back, indicating that ATP had already bound to F1. When stalled at −30° for 5 s (Supplementary Movie 2, right side), F1 rotated back to the original binding angle, indicating that no ATP binding had occurred.

  2. Angle dependence of ATP binding.
    Figure 2: Angle dependence of ATP binding.

    (a) Angle dependence of PON at 60 nM ATP. The 0° represents the original binding angle before manipulation. The stall times were 0.5 s (red), 1 s (blue), 3 s (green) and 5 s (black). Each data point was obtained from 39–536 trials using 13–32 molecules. (b) Time courses of PON. The data in Figure 2a were replotted against stall time: −50° (purple), −30° (cyan), −10° (pink), 0° (black), +10° (blue), +30° (green) and +50° (yellow). Gray line represents the time course in free rotation. The konATP and koffATP were determined by fitting with a single exponential function, {konATP [ATP] / (konATP [ATP] + koffATP)}[1 − exp{–( konATP [ATP] + koffATP)t}], according to the reversible reaction scheme F1 + ATP ⇄ F1ATP. (c) Rate constants determined from the histograms of ATP (red) or GTP (blue) binding dwell in free rotation (circles) for F1 after either an off (triangles) or an on (squares) event. Histograms are given in Supplementary Figures 2 and 4. The red and blue lines represent the rate constants konATP (1.5 × 107 M−1 s−1) and konGTP (4.6 × 106 M−1 s−1), respectively. (df) Angle dependence of kon in d, koff in e and Kd in f. Red and blue symbols represent the values for ATP and GTP determined from Figure 2b and Supplementary Figures 1 and 4. Angle dependences determined by fitting are shown in Table 1. In d, open symbols represent kon in free rotation. Error bars, s.d.

  3. Angle dependence of the hydrolysis step.
    Figure 3: Angle dependence of the hydrolysis step.

    (a) Time course of PON of F1E190D) at 1 mM ATPγS after stalling at −50° (purple), −30° (cyan), 0° (black), +30° (green), +50° (yellow) and +70° (red) from the original catalytic angle; data were fitted as described in Supplementary Methods. Gray line represents the time course in free rotation. Each data point was obtained from 12–102 trials using 4–15 molecules. (b) Angle dependence of khydATPγS (red), ksynATPγS (blue) and koffthioPi (orange). Angle dependences determined by fitting are shown in Table 1. The black circle indicates khydATPγS in free rotation. (c) Angle dependence of the equilibrium constant of hydrolysis, KEHyd, for ATPγS (red) and ATP (blue). (d) Histograms of the catalytic dwell in free rotation (yellow) for F1 after either an off (blue) or an on (red) event. The rate constants were determined to be 0.11 s−1 (yellow), 0.15 s−1 (blue) and 0.12 s−1 (red) by fitting with exponential decay. (e) Time course of an experiment to confirm the reversibility of the hydrolysis step. F1 in a catalytic dwell stalled around +70° for 10 s and then rotated back to the indicated angle to be stalled again for 10 s. Blue points show the period under manipulation. (f) Probability of resynthesis of ATPγS determined experimentally from data in Figure 3e (red) and calculated mathematically from data in Figure 3a (blue). The inset shows a correlation between experimental and expected values determining the efficiency of reversibility (∼95%). Error bars, s.d.

  4. Probability density of rotary angle and rotary potential of pausing F1.
    Figure 4: Probability density of rotary angle and rotary potential of pausing F1.

    (a) The probability densities of the rotary angle in the binding dwell of wild-type F1 at 200 nM ATP (red) and the catalytic dwell of F1E190D) at 1 mM ATP or 1 mM ATPγS (blue). The data were obtained from 6–7 observations for each condition and fitted with Gaussian curves; P(θ) = 3.0 × exp(−θ2 / 345) for ATP binding dwell and P(θ) = 3.0 × exp(−(θ − 80)2 / 323) for hydrolysis dwell. The binding and catalytic angles were assigned as 0° and 80°, respectively. (b) Rotary potentials of F1 in the binding dwell (red) and the catalytic dwell (blue). The rotary potentials were calculated from the probability densities shown in Figure 4a according to Boltzmann's law.

  5. Modulation of kinetic parameters upon γ rotation.
    Figure 5: Modulation of kinetic parameters upon γ rotation.

    (a) Modulation of hydrolysis reactions upon rotation. All data points are plotted along the reaction scheme for one β subunit, and the angles for ATP binding, hydrolysis and Pi (or thioPi) release are assigned as 0°, 200° and 320°, respectively. Red circles represent konATP, blue circles represent khydATPγS, and light and dark green circles represent koffPi and koffthioPi, respectively. Light green triangles represent koffPi at 200°, 240°, 320° and 360° as determined in previous studies16, 18. Solid lines represent the linear regression for konATP (red), khydATPγS (blue) and koffPi (light green). koffPi was only fitted at 320° and 360°. Light orange or light blue lines represent the corrected regressions for konATP and khydATPγS based on the elasticity of the γ subunit (Supplementary Methods). (b) Modulation of elementary steps for synthesis reactions upon rotation. Red, blue and light green represent koffATP, ksynATPγS and konPi, respectively. The light orange line represents the corrected regressions for koffATP based on the elasticity of the γ subunit (Supplementary Methods). (c) Modulation of equilibrium constants upon rotation. Red, blue and light green symbols represent the dissociation constant of ATP (KdATP), the inverse values of the equilibrium constant of ATPγS hydrolysis (1 / KEHyd-ATPγS), and the inverse values of dissociation constant of Pi (1 / KdPi), respectively.

References

  1. Frauenfelder, H., Sligar, S.G. & Wolynes, P.G. The energy landscapes and motions of proteins. Science 254, 15981603 (1991).
  2. Karplus, M. & Kuriyan, J. Molecular dynamics and protein function. Proc. Natl. Acad. Sci. USA 102, 66796685 (2005).
  3. Henzler-Wildman, K. & Kern, D. Dynamic personalities of proteins. Nature 450, 964972 (2007).
  4. Bruice, T.C. & Benkovic, S.J. Chemical basis for enzyme catalysis. Biochemistry 39, 62676274 (2000).
  5. Koshland, D.E. Jr. Ray, W.J. Jr. & Erwin, M.J. Protein structure and enzyme action. Fed. Proc. 17, 11451150 (1958).
  6. Monod, J., Wyman, J. & Changeux, J.P. On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12, 88118 (1965).
  7. Csermely, P., Palotai, R. & Nussinov, R. Induced fit, conformational selection and independent dynamic segments: an extended view of binding events. Trends Biochem. Sci. 35, 539546 (2010).
  8. Boyer, P.D. The ATP synthase—a splendid molecular machine. Annu. Rev. Biochem. 66, 717749 (1997).
  9. Cross, R.L. The rotary binding change mechanism of ATP synthases. Biochim. Biophys. Acta 1458, 270275 (2000).
  10. Yoshida, M., Muneyuki, E. & Hisabori, T. ATP synthase—a marvellous rotary engine of the cell. Nat. Rev. Mol. Cell Biol. 2, 669677 (2001).
  11. Senior, A.E., Nadanaciva, S. & Weber, J. The molecular mechanism of ATP synthesis by F1Fo-ATP synthase. Biochim. Biophys. Acta 1553, 188211 (2002).
  12. Noji, H., Yasuda, R., Yoshida, M. & Kinosita, K. Jr. Direct observation of the rotation of F1-ATPase. Nature 386, 299302 (1997).
  13. Abrahams, J.P., Leslie, A.G., Lutter, R. & Walker, J.E. Structure at 2.8-Å resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621628 (1994).
  14. Yasuda, R., Noji, H., Kinosita, K. Jr. & Yoshida, M. F1-ATPase is a highly efficient molecular motor that rotates with discrete 120 degree steps. Cell 93, 11171124 (1998).
  15. Yasuda, R., Noji, H., Yoshida, M., Kinosita, K. Jr. & Itoh, H. Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase. Nature 410, 898904 (2001).
  16. Adachi, K. et al. Coupling of rotation and catalysis in F1-ATPase revealed by single-molecule imaging and manipulation. Cell 130, 309321 (2007).
  17. Shimabukuro, K. et al. Catalysis and rotation of F1 motor: cleavage of ATP at the catalytic site occurs in 1 ms before 40 degree substep rotation. Proc. Natl. Acad. Sci. USA 100, 1473114736 (2003).
  18. Watanabe, R., Iino, R. & Noji, H. Phosphate release in F1-ATPase catalytic cycle follows ADP release. Nat. Chem. Biol. 6, 814820 (2010).
  19. Itoh, H. et al. Mechanically driven ATP synthesis by F1-ATPase. Nature 427, 465468 (2004).
  20. Rondelez, Y. et al. Highly coupled ATP synthesis by F1-ATPase single molecules. Nature 433, 773777 (2005).
  21. Carter, N.J. & Cross, R.A. Mechanics of the kinesin step. Nature 435, 308312 (2005).
  22. Gebhardt, J.C., Clemen, A.E., Jaud, J. & Rief, M. Myosin-V is a mechanical ratchet. Proc. Natl. Acad. Sci. USA 103, 86808685 (2006).
  23. Gennerich, A., Carter, A.P., Reck-Peterson, S.L. & Vale, R.D. Force-induced bidirectional stepping of cytoplasmic dynein. Cell 131, 952965 (2007).
  24. Iko, Y., Tabata, K.V., Sakakihara, S., Nakashima, T. & Noji, H. Acceleration of the ATP-binding rate of F1-ATPase by forcible forward rotation. FEBS Lett. 583, 31873191 (2009).
  25. Watanabe, R., Iino, R., Shimabukuro, K., Yoshida, M. & Noji, H. Temperature-sensitive reaction intermediate of F1-ATPase. EMBO Rep. 9, 8490 (2008).
  26. Spetzler, D. et al. Microsecond time scale rotation measurements of single F1-ATPase molecules. Biochemistry 45, 31173124 (2006).
  27. Omote, H. et al. The γ-subunit rotation and torque generation in F1-ATPase from wild-type or uncoupled mutant Escherichia coli. Proc. Natl. Acad. Sci. USA 96, 77807784 (1999).
  28. Hirono-Hara, Y., Ishizuka, K., Kinosita, K. Jr., Yoshida, M. & Noji, H. Activation of pausing F1 motor by external force. Proc. Natl. Acad. Sci. USA 102, 42884293 (2005).
  29. Hirono-Hara, Y. et al. Pause and rotation of F1-ATPase during catalysis. Proc. Natl. Acad. Sci. USA 98, 1364913654 (2001).
  30. Sakaki, N. et al. One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar. Biophys. J. 88, 20472056 (2005).
  31. Noji, H. et al. Purine but not pyrimidine nucleotides support rotation of F1-ATPase. J. Biol. Chem. 276, 2548025486 (2001).
  32. Okuno, D., Iino, R. & Noji, H. Stiffness of γ subunit of F1-ATPase. Eur. Biophys. J. 39, 15891596 (2010).
  33. Sielaff, H. et al. Domain compliance and elastic power transmission in rotary FoF1-ATPase. Proc. Natl. Acad. Sci. USA 105, 1776017765 (2008).
  34. Uemura, S. & Ishiwata, S. Loading direction regulates the affinity of ADP for kinesin. Nat. Struct. Biol. 10, 308311 (2003).
  35. Henzler-Wildman, K.A. et al. A hierarchy of timescales in protein dynamics is linked to enzyme catalysis. Nature 450, 913916 (2007).
  36. Ikeguchi, M., Ueno, J., Sato, M. & Kidera, A. Protein structural change upon ligand binding: linear response theory. Phys. Rev. Lett. 94, 078102 (2005).
  37. Shimabukuro, K., Muneyuki, E. & Yoshida, M. An alternative reaction pathway of F1-ATPase suggested by rotation without 80 degrees/40 degrees substeps of a sluggish mutant at low ATP. Biophys. J. 90, 10281032 (2006).
  38. von Ballmoos, C., Cook, G.M. & Dimroth, P. Unique rotary ATP synthase and its biological diversity. Annu. Rev. Biophys. 37, 4364 (2008).
  39. Boyer, P.D., Cross, R.L. & Momsen, W. A new concept for energy coupling in oxidative phosphorylation based on a molecular explanation of the oxygen exchange reactions. Proc. Natl. Acad. Sci. USA 70, 28372839 (1973).
  40. Iino, R., Hasegawa, R., Tabata, K.V. & Noji, H. Mechanism of inhibition by C-terminal alpha-helices of the epsilon subunit of Escherichia coli FoF1-ATP synthase. J. Biol. Chem. 284, 1745717464 (2009).
  41. Kinosita, K. Jr., Adachi, K. & Itoh, H. Rotation of F1-ATPase: how an ATP-driven molecular machine may work. Annu. Rev. Biophys. Biomol. Struct. 33, 245268 (2004).
  42. Nishizaka, T. et al. Chemomechanical coupling in F1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation. Nat. Struct. Mol. Biol. 11, 142148 (2004).
  43. Weber, J., Bowman, C. & Senior, A.E. Specific tryptophan substitution in catalytic sites of Escherichia coli F1-ATPase allows differentiation between bound substrate ATP and product ADP in steady-state catalysis. J. Biol. Chem. 271, 1871118718 (1996).
  44. Shimo-Kon, R. et al. Chemo-mechanical coupling in F1-ATPase revealed by catalytic site occupancy during catalysis. Biophys. J. 98, 12271236 (2010).
  45. Senior, A.E. ATP synthase: motoring to the finish line. Cell 130, 220221 (2007).
  46. Uchihashi, T., Iino, R., Ando, T. & Noji, H. High-speed atomic force microscopy reveals rotary catalysis of rotorless F1-ATPase. Science 333, 755758 (2011).

Download references

Author information

  1. These authors contributed equally to this work.

    • Rikiya Watanabe &
    • Daichi Okuno

Affiliations

  1. Department of Applied Chemistry, School of Engineering, University of Tokyo, Tokyo, Japan.

    • Rikiya Watanabe,
    • Daichi Okuno,
    • Ryota Iino &
    • Hiroyuki Noji
  2. Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan.

    • Shouichi Sakakihara
  3. Department of Biological Science, Florida State University, Tallahassee, Florida, USA.

    • Katsuya Shimabukuro
  4. Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan.

    • Masasuke Yoshida

Contributions

R.W., D.O. and S.S. designed and performed experiments and analyzed data; K.S. gave technical support; R.I. and M.Y. gave technical support and conceptual advice; H.N. designed experiments, conceived the idea behind this paper and wrote this paper with R.W. and R.I.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (935K)

    Supplementary Methods and Supplementary Results

Movies

  1. Supplementary Movie 1 (1M)

    Supplementary Movie 1

  2. Supplementary Movie 2 (2M)

    Supplementary Movie 2

Additional data