| Spastin, a AAA+ ATPase mutated in the neurodegenerative disease hereditary spastic paraplegia, severs microtubules. In examining the mechanism of severing, we find that spastin assembles into a ring-shaped hexamer and loops within the central pore of the hexamer recognize C-terminal amino acids of tubulin. Key pore loop amino acids are required for severing, including one altered by a disease-associated mutation. Spastin also contains a second microtubule binding domain that makes a distinct interaction with microtubules and is required for severing. Because, unlike many AAA+ proteins, spastin has only one target, microtubules, we explored the features of these two spastin-microtubule interactions that might restrict spastin activity to microtubules. The tubulin C-terminal tail and ATP-dependent hexamerization are both important mediators of spastin's affinity for microtubules. The interaction between the tubulin C-terminus and the spastin pore does not require the clustered charged residues that are characteristic of all tubulin isoforms but is mediated by overall negative charge. When the C-terminal 22 amino acids of tubulin are replaced with a very similar acidic unstructured peptide (EB1), spastin binding is retained, whereas it is lost with a less similar sequence. Surprisingly, spastin does not bind EB1-C itself, suggesting spastin also interacts with a region outside the flexible tail of tubulin. The site of this interaction maps to helices 11 and 12 of tubulin. Therefore, the specificity of spastin binding to microtubules is enhanced by multiple sites of contact. Altogether, these data suggest that severing may occur by forces exerted on the C-terminal tail of tubulin, resulting in a conformational change in tubulin that releases it from the microtubule. |
