Functional consequences of preorganized helical structure in the intrinsically disordered cell-cycle inhibitor p27Kip1
Functional consequences of preorganized helical structure in the intrinsically disordered cell-cycle inhibitor p27Kip1
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DOI：
10.1021/bi015763t
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发表时间：
2002-01-22
期刊：
影响因子：
2.9
通讯作者：
Lumb, KJ
中科院分区：
文献类型：
--
作者：
Bienkiewicz, EA;Adkins, JN;Lumb, KJ
p27（Kip 1）通过抑制细胞周期蛋白依赖性激酶（Cdk）活性参与细胞周期调控。当与细胞周期蛋白A-Cdk 2结合时，p27 Cdk抑制结构域具有包括α-螺旋、3（10）螺旋和β-结构的有序构象。相反，未结合的p27 Cdk抑制结构域本质上是无序的（天然未折叠的），如圆二色性光谱所示，缺乏化学位移分散和负异质核Overhauser效应。内在的障碍是不是由于从p27的Cdk抑制结构域的切除，因为全长蛋白质的圆二色光谱也表明了一个很大程度上未折叠的蛋白质。抑制结构域和全长p27作为细胞周期蛋白A-Cdk 2抑制剂是有活性的。使用圆二色性和脯氨酸诱变，我们证明，未结合的p27 Cdk抑制结构域是不完全展开。该结构域含有轻微稳定的螺旋结构，预示着α-螺旋，但不是31（）螺旋，通过结合细胞周期蛋白A-Cdk 2。增加或减少的稳定性的部分预制的α-螺旋在孤立的p27结构域与丙氨酸或脯氨酸取代没有影响形成的p27抑制细胞周期蛋白A-Cdk 2复合物在能量方面。然而，稳定的螺旋与丙氨酸阻碍动力学的抑制复合物的形成，这表明p27从内在的结构紊乱中获得动力学优势。
p27(Kip1) contributes to cell-cycle regulation by inhibiting cyclin-dependent kinase (Cdk) activity. The p27 Cdk-inhibition domain has an ordered conformation comprising an alpha-helix, a 3(10) helix, and beta-structure when bound to cyclin A-Cdk2. In contrast, the unbound p27 Cdk-inhibition domain is intrinsically disordered (natively unfolded) as shown by circular dichroism spectroscopy, lack of chemical-shift dispersion, and negative heteronuclear nuclear Overhauser effects. The intrinsic disorder is not due to the excision of the Cdk-inhibition domain from p27, since circular dichroism spectra of the full-length protein are also indicative of a largely unfolded protein. Both the inhibition domain and full-length p27 are active as cyclin A-Cdk2 inhibitors. Using circular dichroism and proline mutagenesis, we demonstrate that the unbound p27 Cdk-inhibition domain is not completely unfolded. The domain contains marginally stable helical structure that presages the alpha-helix, but not the 31() helix, adopted upon binding cyclin A-Cdk2. Increasing or reducing the stability of the partially preformed alpha-helix in the isolated p27 domain with alanine or proline substitutions did not affect formation of the p27-inhibited cyclin A-Cdk2 complex in energetic terms. However, stabilization of the helix with alanine hindered kinetically the formation of the inhibited complex, suggesting that p27 derives a kinetic advantage from intrinsic structural disorder.