Abstract
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.
Highlights
Spastin is a hexameric microtubule-severing AAA ATPase important for motoneuron integrity
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
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
Summary
Spastin is a hexameric microtubule-severing AAA ATPase important for motoneuron integrity. Clues to spastin’s ATP hydrolysis mechanism come from the primary and tertiary structure and their similarity to those of other AAA ATPases (14) Proteins of this family contain Walker A (or P-loop; motif GXXXGKT) and B motifs (15) in a domain of ϳ220 amino acids with a characteristic consensus pattern (see the Prosite documentation PDOC00572 at the ExPASy Web site). The solution of the three-dimensional structure of nucleotide-free, monomeric spastin by x-ray crystallography (Protein Data Bank code 3B9P) was an important advance (13) It shows that the core AAA module is folded as in other AAA ATPases but extended at its N and C termini and that catalytic key residues are conserved (13). We use quantitative assays of ATP analogs and an inactive mutant to study this problem
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