Structural studies of p21(Waf1/Cip1/Sdi1) in the free and Cdk2-bound state: Conformational disorder mediates binding diversity
Structural studies of p21(Waf1/Cip1/Sdi1) in the free and Cdk2-bound state: Conformational disorder mediates binding diversity
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DOI：
10.1073/pnas.93.21.11504
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发表时间：
1996-10-15
影响因子：
11.1
通讯作者：
Wright, PE
中科院分区：
文献类型：
--
作者：
Kriwacki, RW;Hengst, L;Wright, PE
细胞周期蛋白依赖性激酶 (Cdk) 抑制剂 p21(Waf1/Cip1/Sid1) 对于 p53 依赖性细胞周期控制非常重要，它通过抑制 Cdks 并可能通过抑制 DNA 复制来介导 G(1)/S 阻滞。 Cdk 抑制需要 p21 NH2 末端内大约 60 个氨基酸的序列。我们使用蛋白水解图谱、圆二色性光谱偏振法和核磁共振光谱法证明，作为 Cdk 抑制剂具有活性的 p21 和 NH2 末端片段在游离溶液状态下缺乏稳定的二级或三级结构。然而，与无序游离态形成鲜明对比的是，p21 NH2 末端在与 Cdk2 结合时采用有序稳定构象，如 NMR 光谱直接所示。因此，我们发现 p21 在与其生物靶标之一 Cdk2 结合后发生显着的无序转变。鉴于 p21 能够结合和抑制多种细胞周期蛋白-Cdk 复合物家族（包括细胞周期蛋白 A-Cdk2、细胞周期蛋白 E-Cdk2 和细胞周期蛋白 D-Cdk4），这种结构转变具有深远的意义。我们的研究结果表明，游离 p21 的灵活性或紊乱与结合多样性相关，并为结构紊乱在介导生物系统中的结合特异性中的作用提供了见解。此外，这些观察结果挑战了人们普遍接受的蛋白质观点，即稳定的二级和三级结构是生物活性的先决条件，并表明应在结构-活性关系的背景下考虑更广泛的蛋白质结构观点。
The cyclin-dependent kinase (Cdk) inhibitor p21(Waf1/Cip1/Sid1), important for p53-dependent cell cycle control, mediates G(1)/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.