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

Nature Structural & Molecular Biology volume 24, pages 717–725 (2017)Cite this article

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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.

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Figure 1: Architecture of monomeric and assembled katanin from X-ray diffraction, solution SAXS and cryo-EM structures.
Figure 2: Cryo-EM maps and 3D models for the katanin hexamer in the spiral conformation.
Figure 3: Cryo-EM maps and 3D models for the katanin hexamer in the ring conformation.
Figure 4: Different nucleotide occupancies in the spiral and ring conformations of the katanin hexamer.
Figure 5: Protomer-protomer interface rearrangements between the spiral and ring conformations: transition between tense and relaxed states of the P1 gating protomer.
Figure 6: Pore loop displacement of the gating protomer suggests power stroke for microtubule severing.

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Acknowledgements

We are grateful to N. Grigorieff for initial advice and access to the Krios for collection of one high-resolution data set, R. Diaz-Avalos for data collection advice, J. Hinshaw and H. Wang for TF20 access and F. McNally (University of California, Davis) for a katanin expression plasmid. X-ray diffraction data were collected at beamline 502 of the Advanced Light Source, which is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231. SAXS was performed at Beamline 12ID-B of the Advanced Photon Source, which is a US DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. A.R.-M. is supported by the intramural programs of NINDS and NHLBI.

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Authors and Affiliations

  1. Cell Biology and Biophysics Unit, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, USA

    Elena Zehr, Agnieszka Szyk, Ewa Szczesna & Antonina Roll-Mecak

  2. Biophysics Core, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA

    Grzegorz Piszczek

  3. X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois, USA

    Xiaobing Zuo

  4. Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA

    Antonina Roll-Mecak

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Contributions

E.Z. prepared grids, collected and processed EM data with input from A.R.-M. The high-resolution data set was collected at the Janelia Research Campus (Howard Hughes Medical Institute). All EM data were processed on the Biowulf cluster at the National Institutes of Health. A.R.-M. and E.Z. built and refined models. A.S. purified proteins, obtained crystals, collected X-ray diffraction and SAXS data and performed ATP-binding and ATP-hydrolysis assays. G.P. performed and interpreted AUC. E.W. performed in vitro severing assays. X.Z. collected and processed SAXS data. A.R.-M. refined X-ray structure. A.R.-M. and E.Z. wrote manuscript. All authors reviewed the manuscript. A.R.-M. conceived project and supervised research.

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Correspondence to Antonina Roll-Mecak.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Analytical centrifugation and solution SAXS analyses of C. elegans p60 katanin.

(a)-(c) AUC experimental distribution curves for wild-type katanin (WT) and a Walker B mutant (Glu293Gln) at various concentrations, in the absence or presence of ATP. (d) Agreement between the solution SAXS data (open circle for full-length (FL), open square for ΔMIT katanin) and the DAMMIF models (red solid line for full-length and blue for ΔMIT katanin). (e) Guinier plots for the solution SAXS data in (d). The linear ln[I(q)] versus q2 plot indicates good sample quality. (f) Views of an overlay of a representative model for full-length katanin (green) and the average model obtained using DAMFILT (blue). (g) Top and side views of twenty superimposed bead models of ΔMIT katanin generated from the solution SAXS data. (h) Views of an overlay of a representative model for ΔMIT katanin (gray) and the average model obtained using DAMFILT (magenta). Approximate dimensions shown.

Supplementary Figure 2 Sequence alignment of katanin p60 homologues.

Sequence numbering corresponds to C. elegans katanin p60 (GenBank Accession Code: NP_492257). The N-terminal boundary of the fishhook linker element is approximate and based on the cryo-EM map. α-helices, spirals; β-strands, arrows; random coils, lines, are colored as in Fig. 1e; dashed lines denote unresolved sequences in the X-ray structure. Sequence conservation colored on a gradient from red (100% identity) to yellow (30% identity). Pore loops 1 and 2, ATP interacting elements (Walker A and B) are highlighted by boxes. Black and red ∗ symbols denote inactivating mutations in katanin (Clark-Maguire, S., et al., Genetics, 136, 1994, Pintard, L., et al., Nature, 425, 2003, Clandinin, T. R., et al., Genetics, 134, 1993, McNally, K. P., et al., Molecular biology of the cell, 22, 2011, McNally, K., et al., The Journal of Cell Biology, 175, 2006, McNally, K. P., et al., Journal of Cell Science, 113, 2000, Loughlin, R., et al., Cell, 147, 2011, Whitehead, E., et al., J Mol Biol, 425, 2013) and spastin, respectively (Roll-Mecak, A., et al., Nature, 451, 2008) from previous studies; open circles indicate katanin mutations tested in this study. Alignment generated with Muscle using default settings in Jalview (Waterhouse, A. M., et al., Bioinformatics, 25, 2009).

Supplementary Figure 3 Cryo-EM reconstructions of katanin p60.

(a) Cryo-EM micrograph showing katanin p60 particle distribution in ice and a power spectrum of the micrograph (inset). (b) Selected reference-free 2D class averages. (c) Gold-standard FSC curves for the unmasked and masked cryo-EM reconstructions. (d) Different views of the final sharpened cryo-EM map of katanin p60 in the spiral conformation colored according to the local resolution determined using blocres in Bsoft (Heymann, J. B., J Struct Biol, 133, 2001). (e) Euler distribution plots for the particles contributing to the final reconstruction of the spiral conformation. Red, views containing the highest number of particles. (f) Different views of the final sharpened cryo-EM map of katanin p60 in the ring conformation colored as in (d). (g) Euler distribution plots for the particles contributing to the final reconstruction of the ring conformation.

Supplementary Figure 4 Workflow for classification and refinement for cryo-EM data.

Supplementary Figure 5 Comparison of the cryo-EM katanin hexamer structure and hexamers generated through crystallographic symmetry from monomeric apo structures.

(a) Cryo-EM structure of the katanin hexamer in the spiral conformation (this work); (b) P65 crystal packing of the katanin monomer X-ray crystal structure (this work); (c) P65 crystal packing of D. melanogaster spastin monomer (PDB ID: 3B9P (Roll-Mecak, A., et al., Nature, 451, 2008)); (d) P65 crystal packing of H. sapiens spastin monomer (PDB ID: 3VFD (Taylor, J. L., et al., J Struct Biol, 179, 2012)). Atomic models are colored as in Fig. 2. The models were aligned to each other using protomer P4.

Supplementary Figure 6 Inter-protomer interfaces in the katanin hexamer.

(a) Inter-protomer contacts in the spiral conformation mediated by the α1-α2 and α5-β4 loops, disordered in the X-ray crystal structure of the monomer. P4-P5 interface shown. Cryo-EM map and atomic model colored as in Fig. 2. (b) Close-up view of inter-protomer interactions mediated by helix α12. P4-P5 interface shown. Cryo-EM density presented as transparent gray isosurface. Atomic model is color-coded as in Fig. 1e. The invariant Phe469 and Gly470 highlighted in blue. (c) Side-chains for the arginine finger residues Arg351 and Arg352 at the P2-P3 interface in the cryo-EM map of katanin p60 in the spiral conformation.

Supplementary Figure 7 Different nucleotide occupancies in the spiral and ring conformations of the katanin hexamer.

(a-b) Molecular model with cryo-EM density presented as a transparent gray isosurface showing bound nucleotide at the NBD-HBD junction (Left) and enlarged views of the nucleotide-binding pocket (Right) with the difference map as a transparent gray isosurface of segmented P1 through P6 in the spiral conformation (a) and the ring conformation (b) colored as in Fig. 2. Nucleotide is absent in P1 of the ring conformation, highlighted by the dotted-line oval. Difference maps were calculated between the experimental cryo-EM map and a katanin model-based map without nucleotide in the active site (methods). (c-d) Difference maps corresponding to the gamma-phosphate of ATP are shown as transparent gray isosurfaces for protomers P1 through P6 in the spiral conformation (c) and P2 through P6 in the ring conformation (d). Difference maps were calculated between the experimental cryo-EM map and the katanin model-based map with ADP modeled in the active site (Online methods).

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Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1789 kb)

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Conformational changes between the spiral and ring conformations.

Movie illustrating the katanin structure and changes between the spiral and ring conformations. Movie generated with UCSF Chimera using the Morph Conformation Subroutine. (MOV 62141 kb)

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Zehr, E., Szyk, A., Piszczek, G. et al. Katanin spiral and ring structures shed light on power stroke for microtubule severing. Nat Struct Mol Biol 24, 717–725 (2017). https://doi.org/10.1038/nsmb.3448

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