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Effect of Ca2+ on the microtubule-severing enzyme p60-katanin. Insight into the substrate-dependent activation mechanism

Authors

  • Naoko Iwaya,

    1.  Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan
    2.  Division of Structural Biology, Graduate School of Medicine, Kobe University, Japan
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  • Kohei Akiyama,

    1.  Division of Structural Biology, Graduate School of Medicine, Kobe University, Japan
    2.  Field of Supramolecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, Japan
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  • Natsuko Goda,

    1.  Division of Structural Biology, Graduate School of Medicine, Kobe University, Japan
    2.  Field of Supramolecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, Japan
    3.  Structural Biology Research Center and Division of Biological Science, Nagoya University, Japan
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  • Takeshi Tenno,

    1.  Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan
    2.  Division of Structural Biology, Graduate School of Medicine, Kobe University, Japan
    3.  Structural Biology Research Center and Division of Biological Science, Nagoya University, Japan
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  • Yoshie Fujiwara,

    1.  Division of Structural Biology, Graduate School of Medicine, Kobe University, Japan
    2.  Global Center of Excellence Program for Integrative Membrane Biology, Kobe University, Japan
    3.  Laboratory of Supramolecular Crystallography, Institute for Protein Research, Osaka University, Japan
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  • Daizo Hamada,

    1.  Division of Structural Biology, Graduate School of Medicine, Kobe University, Japan
    2.  Global Center of Excellence Program for Integrative Membrane Biology, Kobe University, Japan
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  • Teikichi Ikura,

    1.  Graduate School of Biomedical Science, Tokyo Medical and Dental University, Japan
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  • Masahiro Shirakawa,

    1.  Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan
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  • Hidekazu Hiroaki

    1.  Division of Structural Biology, Graduate School of Medicine, Kobe University, Japan
    2.  Field of Supramolecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, Japan
    3.  Structural Biology Research Center and Division of Biological Science, Nagoya University, Japan
    4.  Global Center of Excellence Program for Integrative Membrane Biology, Kobe University, Japan
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H. Hidekazu, Structural Biology Research Center and Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan
Fax: +81 52 789 4535
Tel: +81 52 747 6471
E-mail: hiroaki.hidekazu@f.mbox.nagoya-u.ac.jp

Abstract

Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by the p80 subunit (adaptor-p80). p60-katanin consists of an N-terminal domain, followed by a single ATPase associated with various cellular activities (AAA) domain. We have previously shown that the N-terminal domain serves as the binding site for MT, the substrate of p60-katanin. In this study, we show that the same domain shares another interface with the C-terminal domain of adaptor-p80. We further show that Ca2+ ions inhibit the MT-severing activity of p60-katanin, whereas the MT-binding activity is preserved in the presence of Ca2+. In detail, the basal ATPase activity of p60-katanin is stimulated twofold by both MTs and the C-terminal domain of adaptor-p80, whereas Ca2+ reduces elevated ATPase activity to the basal level. We identify the Ca2+-binding site at the end of helix 2 of the N-terminal domain, which is different from the MT-binding interface. On the basis of these observations, we propose a speculative model in which spatial rearrangement of the N-terminal domain relative to the C-terminal AAA domain may be important for productive ATP hydrolysis towards MT-severing. Our model can explain how Ca2+ regulates both severing and ATP hydrolysis activity, because the Ca2+-binding site on the N-terminal domain moves close to the AAA domain during MT severing.

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