title: Spastin's microtubule-binding properties and comparison to katanin.
abstract: Spastin and katanin are ring-shaped hexameric AAA ATPases that sever microtubules, and thus crucially depend on a physical interaction with microtubules. For the first time, we report here the microtubule binding properties of spastin at the single-molecule level, and compare them to katanin. Microscopic fluorescence assays showed that human spastin bound to microtubules by ionic interactions, and diffused along microtubules with a diffusion coefficient comparable to katanin. The microscopic measurement of landing and dissociation rates demonstrated the ionic character of the interaction, which could be mapped to a patch of three lysine residues outside of the catalytic domain of human spastin. This motif is not conserved in Drosophila spastin or katanin, which also bound by non-catalytic parts of the protein. The binding affinities of spastin and katanin were nucleotide-sensitive, with the lowest affinities under ADP,, the highest under ATP-γS conditions. These changes correlated with the formation of higher oligomeric states, as shown in biochemical experiments and electron microscopic images. Vice versa, the artificial dimerization of human spastin by addition of a coiled coil led to a constitutively active enzyme. These observations suggest that dimer formation is a crucial step in the formation of the active complex, and thus the severing process by spastin.
authors.affiliation: Department of Physics E22 Biophysics, Technische Universität München, Garching, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Eckert
authors.forename: Doan Tuong-Van
authors.initials: DT
authors.lastname: Le
authors.forename: Susanne
authors.initials: S
authors.lastname: Link
authors.forename: Lena
authors.initials: L
authors.lastname: Friedmann
authors.forename: Günther
authors.initials: G
authors.lastname: Woehlke
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000818:Animals; D020134:Catalytic Domain; D004058:Diffusion; D004330:Drosophila; D029721:Drosophila Proteins / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007477:Ions; D000074201:Katanin; D008239:Lysine / Q000737:chemistry; D008854:Microscopy, Electron / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000074181:Spastin; D013552:Swine
chemical_list: D029721:Drosophila Proteins; D007477:Ions; D009711:Nucleotides; D049452:Green Fluorescent Proteins; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; C501874:spas protein, Drosophila; D000074181:Spastin; D000074201:Katanin; C400283:SPAST protein, human; D008239:Lysine