|Entry||Database: PDB / ID: 6pek|
|Title||Structure of Spastin Hexamer (Subunit A-E) in complex with substrate peptide|
|Keywords||MOTOR PROTEIN / AAA+ ATPase / Microtubule Severing|
|Function / homology|
Function and homology information
membrane fission / cytokinetic process / nuclear envelope reassembly / positive regulation of microtubule depolymerization / microtubule severing / microtubule-severing ATPase / microtubule-severing ATPase activity / cytoskeleton-dependent cytokinesis / endoplasmic reticulum tubular network / mitotic spindle disassembly ...membrane fission / cytokinetic process / nuclear envelope reassembly / positive regulation of microtubule depolymerization / microtubule severing / microtubule-severing ATPase / microtubule-severing ATPase activity / cytoskeleton-dependent cytokinesis / endoplasmic reticulum tubular network / mitotic spindle disassembly / exit from mitosis / axonal transport of mitochondrion / anterograde axonal transport / microtubule bundle formation / positive regulation of cytokinesis / protein hexamerization / beta-tubulin binding / alpha-tubulin binding / cytoplasmic microtubule organization / metabolic process / lipid droplet / axon cytoplasm / mitotic cytokinesis / isomerase activity / axonogenesis / spindle / midbody / endoplasmic reticulum to Golgi vesicle-mediated transport / protein homooligomerization / cytoplasmic vesicle / microtubule / microtubule binding / nuclear membrane / endosome / ATPase activity / protein-containing complex binding / centrosome / endoplasmic reticulum membrane / perinuclear region of cytoplasm / extracellular exosome / integral component of membrane / nucleoplasm / ATP binding / nucleus / cytosol / cytoplasm
AAA ATPase, AAA+ lid domain / Spastin, chordate / P-loop containing nucleoside triphosphate hydrolase / Spastin / Vps4 oligomerisation, C-terminal / MIT / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / AAA+ ATPase domain
|Biological species||Homo sapiens (human)|
synthetic construct (others)
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å|
|Authors||Han, H. / Schubert, H.L. / McCullough, J. / Monroe, N. / Sundquist, W.I. / Hill, C.P.|
|Funding support|| United States, 1items |
|Citation||Journal: J. Biol. Chem. / Year: 2019|
Title: Structure of spastin bound to a glutamate-rich peptide implies a hand-over-hand mechanism of substrate translocation.
Authors: Han Han / Heidi L Schubert / John McCullough / Nicole Monroe / Michael D Purdy / Mark Yeager / Wesley I Sundquist / Christopher P Hill /
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 ...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 a1A/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, while 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.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
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G: substrate peptide, TYR-GLU-TYR-GLU-TYR-GLU-TYR-GLU
Mass: 54498.328 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPAST, ADPSP, FSP2, KIAA1083, SPG4 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q9UBP0, microtubule-severing ATPase
Mass: 1479.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM
|Has ligand of interest||N|
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: Spastin Hexamer in complex with substrate peptide / Type: COMPLEX / Entity ID: 1,2 / Source: MULTIPLE SOURCES|
|Molecular weight||Experimental value: NO|
|Source (natural)||Organism: Homo sapiens (human)|
|Buffer solution||pH: 8|
|Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Image recording||Electron dose: 57 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)|
|Software||Name: PHENIX / Version: 1.16_3549: / Classification: refinement|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|3D reconstruction||Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 119984 / Symmetry type: POINT|
|Refine LS restraints|
Refinement-ID: ELECTRON MICROSCOPY
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