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首页> 外文期刊>Journal of Molecular Biology >Specific Conformational Dynamics and Expansion Underpin a Multi-Step Mechanism for Specific Binding of p27 with Cdk2/Cyclin A
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Specific Conformational Dynamics and Expansion Underpin a Multi-Step Mechanism for Specific Binding of p27 with Cdk2/Cyclin A

机译：具体的构象动态和扩展基于P27与CDK2 / Cyclin A的特异性结合的多步机制

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摘要

The protein p27, a prominent regulatory protein in eukaryotes and an intrinsically disordered protein (IDP), regulates cell division by causing cell cycle arrest when bound in ternary complex with cyclin-dependent kinase (Cdk2) and cyclins (e.g., Cdk2/Cyclin A). We present an integrative study of p27 and its binding to Cdk2/Cyclin A complex by performing single-molecule multiparameter fluorescence spectroscopy, stoppedflow experiments, and molecular dynamics simulations. Our results suggest that unbound p27 adopts a compact conformation and undergoes conformational dynamics across several orders of magnitude in time (nano-to milliseconds), reflecting a multi-step mechanism for binding Cdk2/Cyclin A. Mutagenesis studies reveal that the region D1 in p27 plays a significant role in mediating the association kinetics, undergoing conformational rearrangement upon initial binding. Additionally, FRET experiments indicate an expansion of p27 throughout binding. The detected local and long-range structural dynamics suggest that p27 exhibits a limited binding surface in the unbound form, and stochastic conformational changes in D1 facilitate initial binding to Cdk2/Cyclin A complex. Furthermore, the post-kinase inhibitory domain (post-KID) region of p27 exchanges between distinct conformational ensembles: an extended regime exhibiting worm-like chain behavior, and a compact ensemble, which may protect p27 against nonspecific interactions. In summary, the binding interaction involves three steps: (i) D1 initiates binding, (ii) p27 wraps around Cdk2/Cyclin A and D2 binds, and (iii) the fully-formed fuzzy ternary complex is formed concomitantly with an extension of the post-KID region. An understanding of how the IDP nature of p27 underpins its functional interactions with Cdk2/Cyclin A provides insight into the complex binding mechanisms of IDPs and their regulatory mechanisms. (C) 2020 The Authors. Published by Elsevier Ltd.
机译：蛋白质p27蛋白，在真核细胞中的显着调节蛋白和固有无序蛋白（IDP），由在三元复合物结合与细胞周期蛋白依赖性激酶（Cdk2的），并且当使细胞周期停滞调节细胞分裂细胞周期蛋白（例如，CDK2 /细胞周期蛋白A） 。我们提出的p27的综合研究，并通过执行其结合到Cdk2 /细胞周期蛋白A复合物单分子多参数荧光光谱学，stoppedflow实验和分子动力学模拟。我们的研究结果表明，未绑定的P27采用跨越几个数量级的一个结构紧凑，经历构象动力学的时间（纳米到毫秒级），反映了结合Cdk2 /细胞周期蛋白A.诱变研究一个多步骤的机制，揭示了区域D1 p27蛋白在介导的关联动力学，在初始结合正在进行的构象重排显著的作用。此外，FRET实验表明p27蛋白的整个结合的膨胀。所检测到的本地和远程的结构动力学表明，p27蛋白表现出未结合的形式的限制结合表面，并且在D1随机的构象变化促进初始结合Cdk2 /细胞周期蛋白A复合物。此外，后激酶抑制结构域（后KID）不同的构象合奏之间的p27交换区域：一个扩展机制表现出蠕虫状链的行为，和紧凑的合奏，其可以保护对p27蛋白的非特异性相互作用。总之，结合相互作用涉及三个步骤：（i）D1发起结合，（ⅱ）围绕Cdk2 /细胞周期蛋白A和D2结合p27的包裹物，和（iii）完全形成的模糊三元复合物与的扩展伴随形成后KID区域。 P27的的IDP性质如何巩固与Cdk2 /细胞周期蛋白A及其功能的相互作用的理解提供了洞察国内流离失所者和其监管机制的复杂的绑定机制。 （c）2020作者。 elsevier有限公司出版

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