Decoding the dynamic mechanism of allosteric activation in the cyclin-dependent kinase Cdk2

项目摘要 该项目旨在探索一种新的研究蛋白激酶构象动力学的方法。 它们的催化活性，并为如何征用蛋白质动力学提供了一个重要的案例研究 用变构小分子来人工控制激酶的活性。 蛋白激酶是一个大的信号蛋白家族，控制细胞的生长和增殖。 真核细胞。激酶的行为就像分子开关，在催化活性的“开”和“开”之间转换。 不活跃的“关闭”状态以严格控制的方式在细胞生理学中带来规定的变化。这 严格的调控控制在癌症中被广泛扰乱，并通过小分子靶向异常的激酶活性。 分子药物现在是许多癌症治疗的重要组成部分。 晶体结构为我们提供了激酶的开启和关闭状态的静态图像，但它们几乎不能告诉我们什么 关于蛋白质如何在这些状态之间转换，这是一个内在的动态过程。的性质 这些动态转变以及它们是如何在疾病中受到干扰的，在很大程度上仍然模糊不清，这一事实已经 阻碍了我们设计变构疗法的能力，这种疗法通过模仿他们的 自然控制机制。相反，现有的激酶抑制剂通过与高度保守的活性结合来发挥作用。 网站，一种使他们缺乏选择性的行动模式。 这个项目的目标是使用实验方法的组合来提供互补的 关于蛋白质动力学的信息，以确定不同构象之间的激酶如何转换 状态，并了解小分子药物如何能够征用激酶动力学来人工 调节蛋白激酶的功能。将核磁共振和光谱学应用于周期蛋白- 依赖的激酶CDK2，细胞周期进程的关键调节因子，我们的目标是揭示1)变构偶联 细胞周期蛋白结合与CDK2激活的联系机制，2)磷酸化如何与细胞周期蛋白协同作用 调节蛋白质动力学和促进催化活性的亚基，以及3)如何动态构象变化 CDK2控制小分子配体进入激酶的变构口袋。这部作品的真知灼见 将从根本上促进我们对蛋白质中变构控制机制的理解，并有助于设定 有效地利用变构来调节激酶的先进变构疗法的设计阶段 功能。

Project Abstract This project reaches towards a new paradigm for how conformational dynamics of protein kinases drive their catalytic activation, and provides an important case study of how protein dynamics can be commandeered to artificially control kinase activity with allosteric small molecules. The protein kinases are a large family of signaling proteins that control cell growth and proliferation in all eukaryotic cells. Kinases behave like molecular switches, transitioning between catalytically active “on” and inactive “off” states in a tightly controlled fashion to bring about prescribed changes in cell physiology. This stringent regulatory control is widely disrupted in cancer, and targeting aberrant kinase activity with small- molecule drugs is now an important component of many cancer treatments. Crystal structures have given us static pictures of the on and off states of kinases, but they tell us little about how the proteins transition between these states, which is an inherently dynamic process. The nature of these dynamic transitions and how they are perturbed in disease remain largely obscure, a fact that has impeded our ability to design allosteric therapeutics that switch kinases to the off state by mimicking their natural control mechanisms. Instead, existing kinase inhibitors work by binding to the highly conserved active site, a mode of action that makes them poorly selective. The goal of this project is to use a combination of experimental methods that provide complementary information about protein dynamics to determine how kinases transition between different conformational states, and to understand how kinase dynamics can be commandeered by small-molecule drugs to artificially modulate kinase function. Using nuclear magnetic resonance and optical spectroscopy applied to the cyclin- dependent kinase Cdk2, a key regulator of cell cycle progression, we aim to reveal 1) the allosteric coupling mechanism that links cyclin binding to activation of Cdk2, 2) how phosphorylation cooperates with the cyclin subunit to tune protein dynamics and promote catalytic activity, and 3) how dynamic conformational changes in Cdk2 control access of small-molecule ligands to allosteric pockets in the kinase. The insights from this work will fundamentally advance our understanding of allosteric control mechanisms in proteins, and help set the stage for the design of advanced allosteric therapeutics that effectively harness allostery to modulate kinase function.