Structural basis for disassembly of katanin heterododecamers

Abstract

The reorganization of microtubules in mitosis, meiosis, and development requires the microtubule-severing activity of katanin. Katanin is a heterodimer composed of an TPasessociated with diverse cellular ctivities (AAA) subunit and a regulatory subunit. Microtubule severing requires ATP hydrolysis by katanin's conserved AAA ATPase domains. Whereas other AAA ATPases form stable hexamers, we show that katanin forms only a monomer or dimers of heterodimers in solution. Katanin oligomers consistent with hexamers of heterodimers or heterododecamers were only observed for an ATP hydrolysis-deficient mutant in the presence of ATP. X-ray structures of katanin's AAA ATPase in monomeric nucleotide-free and pseudo-oligomeric ADP-bound states revealed conformational changes in the AAA subdomains that explained the structural basis for the instability of the katanin heterododecamer. We propose that the rapid dissociation of katanin AAA oligomers may lead to an autoinhibited state that prevents inappropriate microtubule severing or that cyclical disassembly into heterodimers may critically contribute to the microtubule-severing mechanism.

Structural and Molecular Basis for Katanin-Mediated Severing of Glutamylated Microtubules

“…Unlike most AAA ATPases, which exist in oligomeric states without ATP, Kp60 has been reported to be predominantly monomeric and assemble into hexamers in an ATP-and MTdependent manner (Nithianantham et al, 2018;Peng et al, 2013;Roll-Mecak and Vale, 2008;Zehr et al, 2017). However, we found that Kp60AAA shifted from monomeric to hexameric fractions in a concentration-dependent manner, and at high concentrations, both ATP and MT were dispensable for the formation of hexamers ( Figures S1A-S1C).…”

“…However, we found that Kp60AAA shifted from monomeric to hexameric fractions in a concentration-dependent manner, and at high concentrations, both ATP and MT were dispensable for the formation of hexamers ( Figures S1A-S1C). E/Q mutation in the Walker B motif stabilizes hexamer assembly of MT-severing enzymes (Nithianantham et al, 2018;Peng et al, 2013;Roll-Mecak and Vale, 2008;Zehr et al, 2017). We found that the elution profiles of the EQ mutant (Kp60AAA E309Q ) and the wild-type (Kp60AAA WT ) was similar, showing two peaks corresponding to the sizes of a hexamer and a monomer (Figures 1 and S1A-S1D).…”

“…The nucleotide-binding pocket is located at the interface between the two subdomains. With the two helices, a-helix1 (a1) and a12 embracing the NBD at the N and C termini, respectively, the overall structure of the ATPgS-bound human Kp60 is similar to that of other MT-severing enzymes, such as C. elegans MEI-1 (Zehr et al, 2017), human KATNAL1 (Nithianantham et al, 2018), and Drosophila spastin (Roll-Mecak and Vale, 2008;Taylor et al, 2012) ( Figure S1E). Our electron density map explicitly shows the bound ATPgS located at the interface between the HBD and the NBD ( Figure 1D).…”

“…As the apo state of Kp60AAA WT yielded non-diffracting crystals, we compared the structures of the ATPgS-bound Kp60AAA WT and apo Kp60AAA E309Q , which diffracted to 3.0 Å resolution. Kp60AAA E309Q can bind ATP but cannot hydrolyze it and thus traps Kp60AAA in its inactive state (Nithianantham et al, 2018;Peng et al, 2013;Roll-Mecak and Vale, 2008;Zehr et al, 2017). Superimposition of the two Kp60AAA structures reveals that Kp60AAA undergoes a substantial rearrangement upon nucleotide binding ( Figure 1D).…”

Microtubule bundling by MAP65-1 protects against severing by inhibiting the binding of katanin

“…The p80 subunit confers targeting and some degree of regulation of the p60 catalytic activity (Hartman et al, 1998;McNally et al, 2014;Wang et al, 2017). Biochemical and structural studies have shown that the p60 and p80 subunits assemble into hexameric ring structures which interact with the C-terminal tails of tubulin and remove tubulin dimers from microtubule sidewalls to promote severing (McNally and Vale, 1993;Hartman and Vale 1999;Zehr et al, 2017, Nithianantham, et al, 2018Vemu, Szczesna, et al, 2018).…”