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.

Figure 1.. Architecture of monomeric and assembled katanin from X-ray diffraction, solution SAXS and cryo-EM structures.

(a) Top and side views of twenty superimposed ab initio bead models of full-length katanin p60 calculated from solution SAXS data (Supplementary Fig. 1 and Methods). (b) Top view of an overlay of representative models for full-length (grey) and ΔMIT katanin (magenta). Approximate dimensions shown. (c) Pair distance distribution functions for full-length and ΔMIT katanin. (d) Domain diagram of katanin p60; MIT, microtubule interacting and trafficking domain, yellow; flexible linker, pink; AAA domain, grey; fishhook-shaped linker element, light-blue; α1, dark-blue; α12 and 468–472 C-terminal residues, dark-orange. NBD and HBD are highlighted by light green and dark green hatches, respectively as in panel (e). Residue numbers for C. elegans katanin. (e) Cartoon representation of the katanin AAA core crystal structure; N-terminal helix α1, blue; NBD, light green; HBD, dark green; C-terminal helix α12, dark-orange; sulfate ion as stick representation; unresolved residues shown as spheres. (f) Views of the final sharpened 3D density map (13 σ) of the katanin hexamer filtered to 4.4 Å resolution showing a spiral architecture (rotation angles between the different views indicated with arrows). Top, microtubule-binding face. Approximate dimensions shown. Structural elements color-coded as in (d). Bracket indicates open gate between P1 and P6.

Figure 2.. Cryo-EM maps and three-dimensional models for the katanin hexamer in the spiral conformation.

(a) Views of the katanin spiral conformation (rotation angles between the different views indicated with arrows). Protomer P1, green; P2, cyan; P3, blue; P4, orange; P5, purple; P6, red. NBD, HBD, light and dark hue, respectively. Black arrow indicates the open gate between protomers P1 and P6. (b) Top view of the spiral conformation with fitted atomic model. Cryo-EM map, shown as a transparent gray isosurface. The resolution of the map precluded de novo building of the fishhook linker element. Protomers colored as in (a). (c) Views of the atomic models for the katanin spiral conformation in the orientations shown above in panel (a).

Figure 3.. Cryo-EM maps and three-dimensional models for the katanin hexamer in the ring conformation.

(a) Views of the katanin ring conformation (rotation angles between the different views indicated with arrows). Protomer P1, green; P2, cyan; P3, blue; P4, orange; P5, purple; P6, red. NBD, HBD, light and dark hue, respectively. Black arrow indicates the newly formed P1–P6 interface. (b) Top view of the ring conformation with fitted atomic model. Cryo-EM map, shown as a transparent gray isosurface. Protomers colored as in (a). (c) Views of the atomic models for the katanin ring conformation in the orientations shown above in panel (a).

Figure 4.. Different nucleotide occupancies in the spiral and ring conformations of the katanin hexamer.

(a) Superposition of protomers P1 through P6 in the spiral conformation. (Cα overall r.m.s.d. ~ 0.3Å). Protomers colored as in Fig. 2a. (b) P1 structure in the spiral conformation. Bound ATP shown as stick representation. (c-e) Enlarged views of the nucleotide-binding pocket showing bound ATP for protomers P1, P3 and P6 in the spiral conformation with the difference map as transparent gray isosurface (Supplementary Fig. 7a, c and Methods). (f) Superposition of P1, P3 and P6 in the ring conformation showing the 44° rotation of the NBD in the P1 protomer. (g) P1 structure in the ring conformation. Oval highlights ATP-binding region. (h, i) Enlarged views of the nucleotide-binding pocket at two contour levels, 15 σ (h) and 10 σ (i) showing absence of nucleotide in P1. (j) Enlarged view of the nucleotide-binding pocket showing bound ATP for protomer P3 in the ring conformation with the difference map as transparent gray isosurface (Supplementary Fig. 7b, d).

Figure 5.. Protomer-protomer interface rearrangements between the spiral and ring conformations: transition between tense and relaxed states of the P1 gating protomer.

(a, b) Canonical P3–P4 (a) and P1–P2 (b) interfaces in the spiral conformation highlighting the potential salt bridge between invariant Arg244 and Asp322 (Cα shown as spheres). (c) Lack of contacts between the P6 and P1 protomers in the spiral conformation. (d) Canonical P3–P4 interface in the ring conformation highlighting the potential salt bridge between invariant Arg244 and Asp322 (Cα shown as spheres) (e) The relaxed non-canonical P1–P2 interface in the ring conformation. (f) The P6–P1 interface in the ring conformation. Walker B region highlighted by a dashed-line box in all panels and arginine fingers are represented as gray ball and stick (a, b) or Cα spheres (c-f).

Figure 6.. Pore loop displacement of the gating protomer suggests power stroke for microtubule severing.

(a) Superposition of the katanin spiral and ring conformations shows the large movement of the gating protomer P1 (green arrow) and the smaller-scale movement of P6 (red arrow). Protomers are colored as in Fig. 2 in the spiral conformation and are gray in the ring conformation. (b) Cartoon depicting the movement of protomer P1 between the spiral and ring conformations. Protomers colored as in Fig. 2. Arrow highlights the translocation of loop 1 of the P1 gating protomer. (c) Cartoon illustrating proposed power stroke that extracts a tubulin dimer and initiates microtubule lattice breakdown and severing. Left panel, Katanin (blue) assembles as a hexamer with a spiral configuration of the AAA domains and the MIT domains emanating from the AAA motor core and making multivalent interactions with the microtubule (green). The flexible tubulin tail is engaged in the axial pore of the katanin hexamer. Right panel, ATP hydrolysis and release in the gating protomer P1 leads to closure of the AAA ring and a ~20Å displacement in the P1 loop that translocates with it the bound C-terminal tail of a tubulin subunit. The cycle is repeated until lattice contacts unravel and the microtubule severs.