PDBsum entry 3b9p

Hydrolase

PDB id: 3b9p

Contents

Protein chain

268 a.a.

Metals

_CL

Waters ×15

References listed in PDB file

Key reference

Title

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

Authors

A.Roll-Mecak, R.D.Vale.

Ref.

Nature, 2008, 451, 363-367. [DOI no: 10.1038/nature06482]

PubMed id

18202664

Abstract

Spastin, the most common locus for mutations in hereditary spastic paraplegias, and katanin are related microtubule-severing AAA ATPases involved in constructing neuronal and non-centrosomal microtubule arrays and in segregating chromosomes. 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.

Figure 1.
Figure 1: X-ray structure of the nucleotide-free AAA domain of spastin. a, Domain structure of Drosophila spastin: grey, N-terminal domain; red, linker (exon 4, absent in the shorter isoform of spastin used in this study, is hatched); and the AAA domain (coloured according to the X-ray structure). NBD, nucleotide-binding domain; HBD, four-helix bundle domain. Two potential start codons (ATG) are shown (see Supplementary Methods for discussion). The N-terminal boundary of the AAA domain is based on our X-ray structure and differs from that of ref. 14. A segment of the structurally important N-terminal helix of the AAA domain is within what the authors of ref. 14 define as a microtubule-binding domain. The MIT + AAA and AAA constructs are shown schematically below. b, Left, MIT + AAA disassembles the microtubule network when transfected in Drosophila S2 cells and when added to microtubules in vitro, but AAA has no detectable activity at the same concentration (0.15 M). (Weak severing is observed at higher concentrations, Supplementary Fig. 1.) Arrows indicate breaks in microtubules. Scale bar, 5 m. Right, microtubule (MT)-binding and ATPase activities of MIT + AAA and AAA. Microtubule-binding affinity was determined for the Walker B E583Q mutant, which is a stable hexamer and is inactive in severing. c, Ribbon representation of the spastin AAA domain crystal structure. N-terminal helix/loop, magenta; NBD, light green; HBD, dark green; C-terminal helix, blue. The pink sphere depicts a chloride ion. d, Conserved hydrophobic interactions between the N-terminal helix and the main body of the NBD. e, Conserved interactions between the C-terminal helix and the P loop. f, ATPase (red) and microtubule-severing (blue) rates of N- and C-terminal helix mutants. Error bars represent standard errors of the mean (see Methods). WT, wild type. g, Detail of the superposition of spastin and ATP-bound NSF structures^15, showing contacts that keep the N-terminal flap of monomeric spastin (magenta) in an open conformation, unable to stabilize the nucleotide or interact with the neighbouring protomer. Spastin is colour-coded as in panel c. NSF is in grey. Dashed lines, hydrogen bonds.

Figure 4.
Figure 4: Proposed mechanism of severing by spastin and effects of disease mutations. a, Proposed mechanism for microtubule-severing by spastin. The spastin AAA core is shown in cyan with pore loops 1, 2 and 3 highlighted in red and numbered in the figure. The MIT domains are shown as gold ovals. The valency of the interaction of the MIT domains with the microtubule is unknown. On the basis of affinity measurements, it is likely that not all MIT domains are engaged with the microtubule (the potentially unengaged MIT domain is shown hatched). The tubulin heterodimers forming the microtubule are shown in green as a ribbon representation, whereas the C-terminal tubulin tails are shown in red cartoon representation. b, Left, molecular surface of spastin (face A). One protomer is shown in a ribbon representation and residues mutated in HSP patients are shown as violet spheres. Right, in addition to mapping to the pore loops (S589Y, R601L, P631L), disease mutations can interfere with ATP binding (F522C, N527K, K529R) and protomer–protomer interactions (D697N, R704Q, R641C, R601L, P631L). G511R maps to a loop on face A where it could destabilize protomer–protomer interactions and/or the microtubule-binding interface (Supplementary Fig. 4).

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2008, 451, 363-367) copyright 2008.