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

• Published in: The FEBS journal Vol. 279; no. 7; pp. 1339 - 1352
• Main Authors: Iwaya, Naoko, Akiyama, Kohei, Goda, Natsuko, Tenno, Takeshi, Fujiwara, Yoshie, Hamada, Daizo, Ikura, Teikichi, Shirakawa, Masahiro, Hiroaki, Hidekazu
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.04.2012
• Subjects: AAA ATPase; Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - metabolism; Amino Acid Sequence; Animals; Binding Sites; Biochemistry; Calcium; Calcium - metabolism; Enzyme Activation; Enzymes; heteronuclear NMR; Humans; Katanin; Mice; Microtubules - metabolism; microtubule‐severing enzyme; Models, Molecular; molecular modeling; Molecular Sequence Data; Protein Binding; Protein Structure, Tertiary; Sequence Alignment; three‐dimensional domain rearrangement
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2012.08528.x

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. Structured digital •  p80‐CTD binds to p60‐vMIT by pull down (View interaction) •  p80‐CTD and p60‐vMIT bind by nuclear magnetic resonance (View interaction) Here we show that Ca2+ ion binds to the N‐terminal microtubule (MT)‐severing domain of p60‐katanin, MT‐severing enzyme. Ca2+ does not affect the binding between MT and p60‐katanin, whereas microtubule‐dependent ATPase hydrolysis of p60‐katanin was suppressed in the presence of Ca2+. We propose a model that can explain how Ca2+ regulates microtubule‐severing activity of p60‐katanin