Novel binding of the mitotic regulator TPX2 (target protein for Xenopus kinesin-like protein 2) to importin-alpha.
Novel binding of the mitotic regulator TPX2 (target protein for Xenopus kinesin-like protein 2) to importin-alpha.
复制标题
DOI：
10.1074/jbc.m110.102343
复制
发表时间：
2010-06-04
期刊：
影响因子：
--
通讯作者：
Stewart M
中科院分区：
文献类型：
--
作者：
Giesecke A;Stewart M
有丝分裂纺锤体组装的几个方面是由Ran GTdR通过其对纺锤体组装因子和importin-α之间的相互作用的调节来协调的。一个这样的因子是TPX 2，其促进染色体附近的微管组装。TPX 2在与输入蛋白-α结合时受到抑制，后者与输入蛋白-β结合时发生抑制。输入素-α：β相互作用被染色体附近的高RanGTP浓度破坏，释放TPX 2。在更远的区域，Ran主要与GDP结合，TPX 2仍然与输入蛋白-α结合，因此受到抑制。在这里，我们结合使用结构和生化方法来定义TPX 2与输入蛋白-α结合的基础。2.2 nm分辨率的晶体结构显示，TPX 2的主要核定位信号（284 KRKH 287）与importin-α上的次要NLS结合位点结合，该信号已被证明对抑制至关重要。使用互补结合研究（采用与主要或次要NLS结合位点结合受损的输入素-α变体）以及使用主要与主要位点结合的SV 40单组分NLS的竞争试验证实了这种非典型相互作用模式。TPX 2与importin-α结合的不同方式可以解释有丝分裂期间所需的大部分选择性，因为这将减少与其他含NLS的蛋白质结合importin-α的竞争。
Several aspects of mitotic spindle assembly are orchestrated by the Ran GTPase through its modulation of the interaction between spindle assembly factors and importin-α. One such factor is TPX2 that promotes microtubule assembly in the vicinity of chromosomes. TPX2 is inhibited when bound to importin-α, which occurs when the latter is bound to importin-β. The importin-α:β interaction is disrupted by the high RanGTP concentration near the chromosomes, releasing TPX2. In more distal regions, where Ran is predominantly GDP-bound, TPX2 remains bound to importin-α and so is inhibited. Here we use a combination of structural and biochemical methods to define the basis for TPX2 binding to importin-α. A 2.2 Å resolution crystal structure shows that the primary nuclear localization signal (284KRKH287) of TPX2, which has been shown to be crucial for inhibition, binds to the minor NLS-binding site on importin-α. This atypical interaction pattern was confirmed using complementary binding studies that employed importin-α variants in which binding to either the major or minor NLS-binding site was impaired, together with competition assays using the SV40 monopartite NLS that binds primarily to the major site. The different way in which TPX2 binds to importin-α could account for much of the selectivity necessary during mitosis because this would reduce the competition for binding to importin-α from other NLS-containing proteins.