Abstract

Interactions between microtubule (MT) interacting and trafficking (MIT) domains and their binding proteins are important for the accurate progression of many cellular processes that require the AAA+ ATPase machinery. Therefore, knowledge on the structural basis of MIT domain interactions is crucial for understanding the molecular mechanisms underlying AAA+ ATPase function. Katanin is a MT-severing AAA+ ATPase that consists of p60 and p80 subunits. Although, the hexameric p60 subunit is active alone, its association with the p80 subunit greatly enhances both the MT-binding and -severing activities of katanin. However, the molecular mechanism of how the p80 subunit contributes to katanin function is currently unknown. Here, we structurally and functionally characterized the interaction between the two katanin subunits that is mediated by the p60-MIT domain and the p80 C-terminal domain (p80-CTD). We show that p60-MIT and p80-CTD form a tight heterodimeric complex, whose high-resolution structure we determined by X-ray crystallography. Based on the crystal structure, we identified two conserved charged residues that are important for p60-MIT:p80-CTD complex formation and katanin function. Moreover, p60-MIT was compared with other MIT domain structures and similarities are discussed.

Highlights

  • Katanin, spastin and fidgetin are closely related MT-severing enzymes that possess the unique property to catalyse the removal of tubulin heterodimers from the interior of a MT lattice, which results in the breakage of MTs into shorter fragments

  • Current model of MT severing is based on spastin data suggesting that the ring-shaped complexes formed by the AAA+ domains dock onto the MT lattice, which results in an interaction between the positively charged N-terminal pore entrance of the AAA+ ring and the negatively-charged C terminus of tubulin

  • We demonstrated by X-ray crystallography that conserved motifs in ASPM bind at the interface formed by the N- and C-terminal domains of the katanin p60 and p80 subunits, respectively

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Summary

Introduction

Spastin and fidgetin are closely related MT-severing enzymes that possess the unique property to catalyse the removal of tubulin heterodimers from the interior of a MT lattice, which results in the breakage of MTs into shorter fragments. Katanin was shown to localize to spindle poles during mitosis in most animal cells and plays an important role in spindle organization[5,6,7,8,9] It has been identified as a crucial regulator of early embryonic development and recessive mutations or deletions in the katanin-encoding genes are responsible for a dramatic increase in the number of centrosomes and multipolar spindles. The linker and the MIT domains extending from the ring make additional contacts with the MT lattice, thereby increasing the binding of spastin to MTs. The linker and the MIT domains extending from the ring make additional contacts with the MT lattice, thereby increasing the binding of spastin to MTs These additional interactions stabilize the ATP-induced hexameric spastin structure on MTs. According to the proposed model, spastin pulls the C terminus of tubulin through the central pore formed by the hexameric ring of AAA+ domains, thereby generating a mechanical force that destabilizes tubulin–tubulin interactions within the MT lattice and causes MT breakage[11]. The molecular mechanism of how the p80 subunit contributes to katanin function remains to be elucidated

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