Effect of Ca2+ on the microtubule-severing enzyme p60-katanin. Insight into the substrate-dependent activation mechanism.
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 Ca(2+) ions inhibit the MT-severing activity of p60-katanin, whereas the MT-binding activity is preserved in the presence of Ca(2+). In detail, the basal ATPase activity of p60-katanin is stimulated twofold by both MTs and the C-terminal domain of adaptor-p80, whereas Ca(2+) reduces elevated ATPase activity to the basal level. We identify the Ca(2+) -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 Ca(2+) regulates both severing and ATP hydrolysis activity, because the Ca(2+) -binding site on the N-terminal domain moves close to the AAA domain during MT severing.
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto, Japan.
Naoko
N
Iwaya
Kohei
K
Akiyama
Natsuko
N
Goda
Takeshi
T
Tenno
Yoshie
Y
Fujiwara
Daizo
D
Hamada
Teikichi
T
Ikura
Masahiro
M
Shirakawa
Hidekazu
H
Hiroaki
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D000074201:Katanin; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D000251:Adenosine Triphosphatases; D000074201:Katanin; D002118:Calcium