Specific Conformational Dynamics and Expansion Underpin a Multi-Step Mechanism for Specific Binding of p27 with Cdk2/Cyclin A
Specific Conformational Dynamics and Expansion Underpin a Multi-Step Mechanism for Specific Binding of p27 with Cdk2/Cyclin A
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特定的构象动力学和扩展支撑 p27 与 Cdk2/Cyclin A 特异性结合的多步骤机制
DOI：
10.1016/j.jmb.2020.02.010
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发表时间：
2020
影响因子：
5.6
通讯作者：
Sanabria, Hugo
中科院分区：
文献类型：
--
作者：
Tsytlonok, Maksym;Hemmen, Katherina;Hamilton, George;Kolimi, Narendar;Felekyan, Suren;Seidel, Claus A.M.;Tompa, Peter;Sanabria, Hugo
P27蛋白是真核生物中一种重要的调控蛋白，也是一种固有的无序蛋白(IDP)，当它与细胞周期蛋白依赖性蛋白2(CDK2)和细胞周期蛋白A(CDK2/Cyclin A)三元复合体结合时，通过引起细胞周期停滞来调节细胞分裂。我们通过单分子多参数荧光光谱、停流实验和分子动力学模拟对p27及其与CDK2/Cyclin A复合体的结合进行了综合研究。我们的结果表明，未结合的p27采用了紧凑的构象，并在几个数量级的时间内(纳秒到毫秒)经历了构象动力学，反映了CDK2/Cyclin A结合的多步骤机制。突变研究表明，p27中的d1区域在调节结合动力学中起着重要作用，在初始结合时进行构象重排。此外，FRET实验表明p27在整个结合过程中都有扩展。检测到的局部和远程结构动力学表明，p27以未结合的形式显示出有限的结合面，并且D1中的随机构象变化促进了最初与CDK2/Cyclin A复合体的结合。此外，p27的激酶后抑制域(POST-KID)区域在不同的构象集合之间交换：呈现蠕虫样链行为的扩展区域和可能保护p27免受非特异性相互作用的紧凑集合。综上所述，结合作用涉及三个步骤：(I)D1启动结合，(Ii)p27包裹在CDK2/Cyclin A和D2结合周围，以及(Iii)完全形成的模糊三元复合体伴随着后-KID区域的延伸而形成。了解p27的IDP性质如何支持其与CDK2/Cyclin A的功能相互作用有助于深入了解IdP的复杂结合机制及其调控机制。
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, stopped-flow 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.
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影响因子：
3.3
作者：
Shirvanyants, David;Ding, Feng;Tsao, Douglas;Ramachandran, Srinivas;Dokholyan, Nikolay V.
通讯作者：
Dokholyan, Nikolay V.
DOI：
10.1002/cphc.201100897
发表时间：
2012-03
期刊：
Chemphyschem : a European journal of chemical physics and physical chemistry
影响因子：
--
作者：
Felekyan S;Kalinin S;Sanabria H;Valeri A;Seidel CA
通讯作者：
Seidel CA
影响因子：
2.9
作者：
Bienkiewicz, EA;Adkins, JN;Lumb, KJ
通讯作者：
Lumb, KJ
影响因子：
5.6
作者：
S. Sivakolundu;D. Bashford;R. Kriwacki
通讯作者：
S. Sivakolundu;D. Bashford;R. Kriwacki
DOI：
10.1016/b978-0-12-816463-1.00006-7
发表时间：
2019
期刊：
Spectroscopy and Dynamics of Single Molecules
影响因子：
--
作者：
George L Hamilton;H. Sanabria
通讯作者：
George L Hamilton;H. Sanabria