Abstract
Many members of the AAA+ ATPase family function as hexamers that unfold their protein substrates. These AAA unfoldases include spastin, which plays a critical role in the architecture of eukaryotic cells by driving the remodeling and severing of microtubules, which are cytoskeletal polymers of tubulin subunits. Here, we demonstrate that a human spastin binds weakly to unmodified peptides from the C-terminal segment of human tubulin α1A/B. A peptide comprising alternating glutamate and tyrosine residues binds more tightly, which is consistent with the known importance of glutamylation for spastin microtubule severing activity. A cryo-EM structure of the spastin-peptide complex at 4.2 Å resolution revealed an asymmetric hexamer in which five spastin subunits adopt a helical, spiral staircase configuration that binds the peptide within the central pore, whereas the sixth subunit of the hexamer is displaced from the peptide/substrate, as if transitioning from one end of the helix to the other. This configuration differs from a recently published structure of spastin from Drosophila melanogaster, which forms a six-subunit spiral without a transitioning subunit. Our structure resembles other recently reported AAA unfoldases, including the meiotic clade relative Vps4, and supports a model in which spastin utilizes a hand-over-hand mechanism of tubulin translocation and microtubule remodeling.
Highlights
Many members of the AAA؉ ATPase family function as hexamers that unfold their protein substrates. These AAA unfoldases include spastin, which plays a critical role in the architecture of eukaryotic cells by driving the remodeling and severing of microtubules, which are cytoskeletal polymers of tubulin subunits
Spastin belongs to the meiotic clade of AAA unfoldases that includes katanin, which shares spastin’s microtubule-severing activity, and Vps4, which performs an analogous activity against ESCRT-III filaments [3]
The class II pockets are created by pore loop 1 Val-416 residues of adjacent subunits and are flanked by pore loop 2 His-455 residues of the first and preceding subunit and by Arg-460 of the first subunit. These pockets bind the peptide side chains, with residues 2, 4, 6, and 8 fitting into class I pockets and residues 3, 5, 7, and 9 into class II pockets. (Note that residue number 1 of the shorter Vps4-bound peptide [13] corresponds to residue 2 of (EY)5.) The density does not distinguish between peptide glutamate and tyrosine side chains, the positioning of glutamates in class II pockets is consistent with the presence of His-455, which is conserved in spastins and katanins, and is the only potentially charged residue at the binding surface that differs between the microtubule-severing AAA unfoldases and their meiotic clade relative Vps4, where it is a serine residue
Summary
Human spastin is expressed from a single gene as four different isoforms [26]. The full-length (M1) protein comprises 616 amino acid residues, whereas use of an alternative initiator methionine produces the more abundant M87 isoforms [27, 28]. Bound spastin with a KD of ϳ30 M, whereas the C-terminal peptide bound even more weakly, with a KD in excess of 100 M (Fig. 1) These data are consistent with the finding that the interaction of spastin with tubulin C-terminal residues is functionally important but inherently weak [31], and that posttranslational modifications enhance the microtubule-severing activity of spastin [8]. E8 binding was weak, whereas (EY) bound with a KD of 0.3 Ϯ 0.1 M (Fig. 1), which is similar to that of an ESCRT-III– derived peptide binding to Vps4 [11] This relatively tight binding affinity may reflect the importance of an intermediate level of posttranslational glutamylation for optimal tubulin severing [8], and made (EY) an attractive target for determination of a spastin-peptide complex structure. In contrast to the tightlypacked interfaces that define the helical assembly of subunits A–E (Fig. 3A), the EF and FA interfaces are relatively open at the nucleotide-binding sites in the large domain, as if to allow for nucleotide exchange (Fig. 3, B and C)
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