Cooperative Interactions in the Microtubule-Severing AAA ATPase Spastin

Abstract

Spastin is a hexameric ring AAA ATPase that severs microtubules. To see how the ring complex funnels the energy of one or multiple ATP hydrolysis events to the site of mechanical action, we investigate here the cooperativity of spastin. Several lines of evidence show that two neighbor subunits interact strongly with each other: (i) the ATP-activated ATPase activity shows a Hill coefficient of ∼2; (ii) inactive mutant subunits and (iii) non-hydrolyzable ATP analogs inhibit the activity of spastin in a hyperbolic dependence, characteristic for two interacting species. A quantitative model fits the data well, and strongly disfavors orders of cooperativity higher than 2. These observations are relevant for patients suffering from SPG4-type hereditary spastic paraplegia, and can explain why single amino acid exchanges lead to dominant-negative phenotypes. In severing assays, wildtype spastin is even more sensitive towards the presence inactive mutants than in enzymatic assays, suggesting a weak coupling of ATPase and severing activity. Together, these observations indicate that each of spastin's six catalytic sites depends on the presence of an active neighbor site, and that ATP hydrolysis in all subunits is required for full severing activity.