Structures of the CDK12/CycK complex with AMP-PNP reveal a flexible C-terminal kinase extension important for ATP binding.
Structures of the CDK12/CycK complex with AMP-PNP reveal a flexible C-terminal kinase extension important for ATP binding.
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DOI：
10.1038/srep17122
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发表时间：
2015-11-24
影响因子：
4.6
通讯作者：
Bullock AN
中科院分区：
文献类型：
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作者：
Dixon-Clarke SE;Elkins JM;Cheng SW;Morin GB;Bullock AN
细胞周期蛋白依赖性激酶12(CDK12)通过磷酸化RNA聚合酶II的C末端结构域(CTD)促进转录延长。结构-功能研究表明，这种活性依赖于C末端的激酶延伸，以及细胞周期蛋白K(CycK)的结合。为了更好地定义这些相互作用，我们测定了在AMP-PNP存在下，有和没有激酶延伸的人CDK12/CycK复合体的晶体结构。这些结构揭示了CDK的新特征，包括一个大的β4-β5环插入，这有助于与细胞周期蛋白的N叶相互作用。我们还观察到了两种不同构象的C-末端激酶延伸，它们有效地打开和关闭了ATP口袋。最值得注意的是，只有在闭合状态下才能观察到结合的AMP-PNP。该C-末端结构的截断也降低了AMP-PNP的结合，以及CDK12/CycK复合体的催化活性。进一步的动力学测量表明，全长CDK12/CycK复合体的活性明显高于两个结晶构建体，这表明额外的结构域具有关键作用。总体而言，这些结果证明了CDK12中C末端延伸的内在灵活性，并强调了它对ATP结合和激酶活性的重要性。
Cyclin-dependent kinase 12 (CDK12) promotes transcriptional elongation by phosphorylation of the RNA polymerase II C-terminal domain (CTD). Structure-function studies show that this activity is dependent on a C-terminal kinase extension, as well as the binding of cyclin K (CycK). To better define these interactions we determined the crystal structure of the human CDK12/CycK complex with and without the kinase extension in the presence of AMP-PNP. The structures revealed novel features for a CDK, including a large β4-β5 loop insertion that contributes to the N-lobe interaction with the cyclin. We also observed two different conformations of the C-terminal kinase extension that effectively open and close the ATP pocket. Most notably, bound AMP-PNP was only observed when trapped in the closed state. Truncation of this C-terminal structure also diminished AMP-PNP binding, as well as the catalytic activity of the CDK12/CycK complex. Further kinetic measurements showed that the full length CDK12/CycK complex was significantly more active than the two crystallised constructs suggesting a critical role for additional domains. Overall, these results demonstrate the intrinsic flexibility of the C-terminal extension in CDK12 and highlight its importance for both ATP binding and kinase activity.