Katanin spiral and ring structures shed light on power stroke for microtubule severing

Abstract

Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases important for the biogenesis and maintenance of complex microtubule arrays in axons, spindles and cilia. Because of a lack of known 3D structures for these enzymes, their mechanism of action has remained poorly understood. Here we report the X-ray crystal structure of the monomeric AAA katanin module from Caenorhabditis elegans and cryo-EM reconstructions of the hexamer in two conformations. The structures reveal an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to microtubule-severing enzymes and critical for their function. The reconstructions show that katanin cycles between open spiral and closed ring conformations, depending on the ATP occupancy of a gating protomer that tenses or relaxes interprotomer interfaces. Cycling of the hexamer between these conformations would provide the power stroke for microtubule severing.

Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

“…These structures show how asymmetric hexameric rings of ClpX dock with symmetric heptameric rings of ClpP and reveal how the pore-1, pore-2 and RKH loops of ClpX function in substrate binding. ClpX adopts a spiral conformation, with neighboring pore-1 loops interacting with every two residues of substrate in the axial channel, as observed in other AAA+ unfolding and remodeling machines (Puchades et al, 2017;de la Peña et al, 2018;Dong et al, 2019;Majumder et al, 2019;Ripstein et al, 2017;Gates et al, 2017;Zehr et al, 2017;Han et al, 2017;Su et al, 2017;Sun et al, 2017;Yu et al, 2018;White et al, 2018;Cooney et al 2019;Rizo et al, 2019;Shin et al, 2019;Twomey et al, 2019). Based on these structural features, strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been proposed.…”

“…Step). Based on similar hexamer architectures and/or substrate interactions, a SC/2R translocation model has also been proposed for Lon, for PAN/20S, and for the AAA+ proteinremodeling machines ClpB/Hsp104, CDC48/p97, NSF, and Vps4 (Gates et al, 2017;Han et al, 2017;Ripstein et al, 2017;Su et al, 2017;Sun et al, 2017;Zehr et al, 2017;Yu et al, 2018;White et al, 2018;Cooney et al 2019;Majumder et al, 2019;Rizo et al, 2019;Twomey et al, 2019;Shin et al, 2019).…”

Structural basis for disassembly of katanin heterododecamers

“…In contrast, each of these proteins has been reported to form a stable hexamer at low concentration when ATP hydrolysis is blocked by the mutation of a con-served Glu (E) in the Walker B motif to Gln (Q) (17)(18)(19)(20)(21). Structural studies have revealed that the ATP hydrolysis-deficient Walker B mutant of p60 katanin can form a right-handed hexameric spiral with all six protomers bound by ATP or a closed hexameric ring with five ATP-bound protomers and one nucleotide-free protomer (22). However, earlier fluorescence resonance energy transfer (FRET) experiments indicate that oligomers of WT p60 katanin could be detected only in the presence of both a microtubule substrate and a nonhydrolyzable ATP analog (19).…”

Structural asymmetry governs the assembly and GTPase activity of McrBC restriction complexes

“…Our structural analysis reveals that TgMcrB AAA forms an asymmetric hexamer, similar to the architecture adopted by many other AAA+ family proteins (de la Pena et al, 2018; Enemark and Joshua-Tor, 2006; Gates et al, 2017; Puchades et al, 2017; Ripstein et al, 2017; Twomey et al, 2019; Zehr et al, 2017; Zhao et al, 2015). Asymmetry appears to be maintained by the conformation of key interface residues – Arg360, Glu527 and Tyr530 in one monomer and Arg414, Asp420 and Arg424 in its neighbor – acting in trans .…”