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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.
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