Subunit Interactions and cooperativity in the microtubule-severing AAA ATPase spastin.
Spastin is a hexameric ring AAA ATPase that severs microtubules. To see whether the ring complex funnels the energy of multiple ATP hydrolysis events to the site of mechanical action, we investigate here the cooperativity of spastin. Several lines of evidence indicate that interactions among two subunits dominate the cooperative behavior: (i) the ATPase activity shows a sigmoidal dependence on the ATP concentration; (ii) ATPγS displays a mixed-inhibition behavior for normal ATP turnover; and (iii) inactive mutant subunits inhibit the activity of spastin in a hyperbolic dependence, characteristic for two interacting species. A quantitative model based on neighbor interactions fits mutant titration experiments well, suggesting that each subunit is mainly influenced by one of its neighbors. These observations are relevant for patients suffering from SPG4-type hereditary spastic paraplegia and explain why single amino acid exchanges lead to a dominant negative phenotype. In severing assays, wild type spastin is even more sensitive toward the presence of inactive mutants than in enzymatic assays, suggesting a weak coupling of ATPase and severing activity.
Department of Physics E22 (Biophysics), Technische Universität München, James-Franck-Strasse 1, D-85748 Garching/Munich, Germany.
Thomas
T
Eckert
Susanne
S
Link
Doan Tuong-Van
DT
Le
Jean-Philippe
JP
Sobczak
Anja
A
Gieseke
Klaus
K
Richter
Günther
G
Woehlke
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000465:Algorithms; D000818:Animals; D057075:Enzyme Assays; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008870:Microtubules / Q000737:chemistry; D020125:Mutation, Missense; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry*; D017384:Sequence Deletion; D000074181:Spastin; D013552:Swine; D059008:Time-Lapse Imaging
D021122:Protein Subunits; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000074181:Spastin; C400283:SPAST protein, human