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Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin

Abstract

Spastin, the most common locus for mutations in hereditary spastic paraplegias1, and katanin are related microtubule-severing AAA ATPases2,3,4,5,6 involved in constructing neuronal7,8,9,10 and non-centrosomal7,11 microtubule arrays and in segregating chromosomes12,13. The mechanism by which spastin and katanin break and destabilize microtubules is unknown, in part owing to the lack of structural information on these enzymes. Here we report the X-ray crystal structure of the Drosophila spastin AAA domain and provide a model for the active spastin hexamer generated using small-angle X-ray scattering combined with atomic docking. The spastin hexamer forms a ring with a prominent central pore and six radiating arms that may dock onto the microtubule. Helices unique to the microtubule-severing AAA ATPases surround the entrances to the pore on either side of the ring, and three highly conserved loops line the pore lumen. Mutagenesis reveals essential roles for these structural elements in the severing reaction. Peptide and antibody inhibition experiments further show that spastin may dismantle microtubules by recognizing specific features in the carboxy-terminal tail of tubulin. Collectively, our data support a model in which spastin pulls the C terminus of tubulin through its central pore, generating a mechanical force that destabilizes tubulin–tubulin interactions within the microtubule lattice. Our work also provides insights into the structural defects in spastin that arise from mutations identified in hereditary spastic paraplegia patients.

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Figure 1: X-ray structure of the nucleotide-free AAA domain of spastin.
Figure 2: Model of active, hexameric spastin from light and small-angle X-ray scattering.
Figure 3: Role of the tubulin C terminus and the spastin pore in microtubule-severing.
Figure 4: Proposed mechanism of severing by spastin and effects of disease mutations.

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Protein Data Bank

Data deposits

Atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank under the accession number 3B9P.

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Acknowledgements

We thank C. Ralston for access to beamlines at the Advanced Light Source (Lawrence Berkeley National Laboratory), G. Hura for assistance during the SAXS experiments and data processing, N. Zhang for assistance with molecular biology, D. Southword for advice with the static multi-angle scattering experiments, T. Huckaba for the anti-Glu α-tubulin antibody, and H. Bourne and A. Ferre-D’Amare for support and critical reading of the manuscript. R.D.V. is a Howard Hughes Medical Institute investigator. A.R.-M. has received support from the Damon Runyon Cancer Research Foundation, the NIH and the Burroughs Wellcome Fund.

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Correspondence to Ronald D. Vale.

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The file contains Supplementary Figures 1-8 and Legends, Supplementary Methods and Supplementary Tables 1-2 (PDF 3427 kb)

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Roll-Mecak, A., Vale, R. Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin. Nature 451, 363–367 (2008). https://doi.org/10.1038/nature06482

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