Spastin's microtubule-binding properties and comparison to katanin.
Spastin's microtubule-binding properties and comparison to katanin.
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DOI：
10.1371/journal.pone.0050161
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Woehlke G
中科院分区：
文献类型：
--
作者：
Eckert T;Le DT;Link S;Friedmann L;Woehlke G
Spastin 和 katanin 是环状六聚体 AAA ATP 酶，可切断微管，因此关键取决于与微管的物理相互作用。我们首次在单分子水平上报告了 spastin 的微管结合特性，并将其与 katanin 进行了比较。显微荧光测定表明，人spastin通过离子相互作用与微管结合，并沿着微管扩散，其扩散系数与katanin相当。着陆和解离速率的显微测量证明了相互作用的离子特征，该特征可以映射到人 spastin 催化结构域之外的三个赖氨酸残基的补丁。该基序在果蝇 spastin 或 katanin 中并不保守，它们也与蛋白质的非催化部分结合。 spastin和katanin的结合亲和力是核苷酸敏感的，在ADP条件下亲和力最低，在ATP-γS条件下最高。正如生化实验和电子显微镜图像所示，这些变化与更高寡聚状态的形成相关。反之亦然，通过添加卷曲线圈对人 spastin 进行人工二聚化，产生组成型活性酶。这些观察结果表明，二聚体形成是活性复合物形成的关键步骤，因此是 spastin 切断过程的关键步骤。
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.
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