Structure of spastin bound to a glutamate-rich peptide implies a hand-over-hand mechanism of substrate translocation

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.

Atomistic Basis of Force Generation, Translocation, and Coordination in a Viral Genome Packaging Motor

“…The DNA-gripping residues of asccφ28 are the same as depicted in Fig. 4B. (sub)domains (Han et al, 2020;Zehr et al, 2020). Indeed, our MD simulation of the ATP-bound asccφ28 ATPase domain assembly showed a conformational change from the planar ring assembly to a helical assembly as the subunits shifted to associate with the helical phosphate backbone of substrate DNA (Fig.…”

Cutting, Amplifying, and Aligning Microtubules with Severing Enzymes

“…Hexamer Assembly on the Microtubule Lattice Katanin and spastin have low oligomerization tendencies in the absence of microtubules and the predominant forms in solution are most likely to be monomeric [28][29][30]. While spastin, katanin, and fidgetin hexamers have been observed in solution, the proteins are either ATPase defective or truncated [30][31][32] or bound to non-hydrolyzable ATP analogs at high-micromolar protein concentrations [33,34]. However, oligomers are readily seen by single-molecule fluorescence and Förster resonance energy transfer on the microtubule lattice [28,35,36].…”

“…Thus, the newly created microtubule will act as a seed to increase the total microtubule number and mass ( Figure IC). in an ATP-like conformation [31,37], while a spastin structure with ADP-beryllium fluoride (ADP-BeF x ) shows a 'broken spiral' in which the sixth AAA (magenta in Figure 1D) is in the apo state and has moved up to form a closed structure ( Figure 1C,D) [33].…”

Spastin recovery in hereditary spastic paraplegia by preventing neddylation-dependent degradation

“…In neurons, spastin has key roles in axonal transport and regeneration ( 4 , 5 , 6 ). This enzyme functions as hexamers that drive the remodeling and severing of MT by tugging the C-terminal tail of tubulin through the central pore of the hexamer ( 7 , 8 , 9 ). Thus far, four spastin isoforms have been identified, M1 (68 kD), a shorter isoform lacking the first 86 aa (M87, 60 kD), and splice variants of both of these, excluding exon 4 (M1∆4, 64 kD, and M87∆4, 55 kD).…”