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Cooperative three-step motions in catalytic subunits of F1-ATPase correlate with 80° and 40° substep rotations

Nature Structural & Molecular Biology volume 15, pages 1326–1333 (2008)Cite this article

Abstract

Rotation of the central shaft γ subunit in a molecular motor F1-ATPase is assumed to correlate with and probably be driven by domain motions of the three catalytic β subunits. Here we observe directly these β motions through an attached fluorophore, concomitantly with 80° and 40° substep rotations of γ in the same single molecules. We show the sequence of conformations that each β subunit undergoes in three-step bending, a ∼40° counterclockwise turn followed by two ∼20° clockwise turns, occurring in synchronization with two substep rotations of γ. The results indicate that most previous crystal structures mimic the conformational set of three β subunits in the catalytic dwells. Moreover, a previously undescribed set of β conformations, open, closed and partially closed, is revealed in the ATP-waiting dwells. The present study thus bridges the gap between the chemical and mechanical steps in F1-ATPase.

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Figure 1: Simultaneous observation of motions in β and rotation of γ in single-molecule α3β3γ subcomplex of F1-ATPase.
Figure 2: The β subunit shows angular motions upon rotational substeps of γ.
Figure 3: Conformations of β at 80° and 320° of γ revealed by a complementary hybrid species.
Figure 4: Three β subunits of F1 are in C′CO conformation during ATP-waiting dwells and CCO conformation during catalytic dwells as a result of domain motions.
Figure 5: The three-step cooperative motion of β subunits in driving two rotational substeps of γ.

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Acknowledgements

We are grateful to S. Kimura for technical assistance, T. Amano for advice on preparation and biochemical studies of the β subunit, H. Noji, T. Ariga, R. Iino, H. Ueno, K. Adachi and M. Takeda for discussion on the rotation assay, J. Corrie for discussion on fluorescent probes, Y. Matsumoto and T. Shibahara for advice on MS, S. Hayashi and M. Ikeguchi for discussion on crystal structures, A.P. Dominey for critically reading the manuscript, J. Yajima for discussion, K. Abe for the objective lens and our colleagues for discussion, technical advice and assistance. This study was supported in part by the ERATO Yoshida ATP System Project of Japan Science and Technology Agency, a Grant-in-Aid for Scientific Research on Priority Areas (No. 18074008 to T.N. and T.M.), a Grant-in-Aid for Young Scientists (B) (No.18770138 to T.M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and from the Japan Society for the Promotion of Science, Saneyoshi Scholarship (No.1817 to T.M.), the Grant Program for Natural Science from the Asahi Glass Foundation to T.M. and a grant from the New Energy and Industrial Technology Development Organization (NEDO) to T.N.

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Authors and Affiliations

  1. Department of Physics, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, 171-8588, Tokyo, Japan

    Tomoko Masaike, Fumie Koyama-Horibe & Takayuki Nishizaka

  2. Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, 588-2 Iwaoka, Nishi-ku, 651-2492, Kobe, Japan

    Kazuhiro Oiwa

  3. Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, 226-8503, Yokohama, Japan

    Masasuke Yoshida

  4. ICORP ATP Synthesis Regulation Project, Japan Science and Technology Agency, National Museum of Emerging Science and Innovation, 2-41 Aomi, Koto-ku, 135-0064, Tokyo, Japan

    Masasuke Yoshida

  5. PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi City, 332-0012, Saitama, Japan

    Takayuki Nishizaka

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  1. Tomoko Masaike
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  2. Fumie Koyama-Horibe
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  3. Kazuhiro Oiwa
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Contributions

T.M., F.K.-H. and T.N. performed the experiments; K.O., M.Y. and T.N. supervised the project; T.M. wrote the manuscript.

Corresponding authors

Correspondence to Masasuke Yoshida or Takayuki Nishizaka.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 (PDF 1250 kb)

Supplementary Video 1

The video shows motions of the C-terminal domain in a single catalytic subunit β upon rotational substeps of the γ-shaft in F1-ATPase shown in Fig. 2b-f. A fluorophore linked to β (right column) and polystyrene beads attached to γ (left column) are simultaneously observed. In different angles of γ and chemical states of β (0°, 120°, 200° and 240° from top to bottom rows), the fluorophore shows phase shifts in spite of synchronized polarization of the excitation laser. It indicates domain motions of β represented by angular motions of the C-terminal helix upon rotational substeps of γ. Colored boxes show peak frames obtained by sinusoidal fitting of fluorescence intensity during each dwell. Images were recorded at 30 frames per second, and are played at 5 frames per second (1/6 speed) for 45 frames (3 cycles of polarization of the excitation laser). The images of beads were rotated with bilinear interpolation so that the beads point to the 12 o'clock direction at the 0° dwell. All contrast manipulations and enhancements for clarity were strictly linear in each column. Frame sizes are 0.57 μm × 0.52 μm (height × width) for the beads window (left frames, inner size) and 0.97 μm × 0.97 μm (height × width) for the fluorescence window (right frames, inner size). To see the phase shift of the fluorescence intensity clearly, it is recommended to loop the movie and play continuously. (MOV 769 kb)

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Masaike, T., Koyama-Horibe, F., Oiwa, K. et al. Cooperative three-step motions in catalytic subunits of F1-ATPase correlate with 80° and 40° substep rotations. Nat Struct Mol Biol 15, 1326–1333 (2008). https://doi.org/10.1038/nsmb.1510

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