Sohn Jungsan, Parks Jerry M, Buhrman Gregory, Brown Paul, Kristjánsdóttir Kolbrun, Safi Alexias, Edelsbrunner Herbert, Yang Weitao, Rudolph Johannes
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Biochemistry. 2005 Dec 20;44(50):16563-73. doi: 10.1021/bi0516879.
Cdc25 phosphatases are key activators of the eukaryotic cell cycle and compelling anticancer targets because their overexpression has been associated with numerous cancers. However, drug discovery targeting these phosphatases has been hampered by the lack of structural information about how Cdc25s interact with their native protein substrates, the cyclin-dependent kinases. Herein, we predict a docked orientation for Cdc25B with its Cdk2-pTpY-CycA protein substrate by a rigid-body docking method and refine the docked models with full-scale molecular dynamics simulations and minimization. We validate the stable ensemble structure experimentally by a variety of in vitro and in vivo techniques. Specifically, we compare our model with a crystal structure of the substrate-trapping mutant of Cdc25B. We identify and validate in vivo a novel hot-spot residue on Cdc25B (Arg492) that plays a central role in protein substrate recognition. We identify a hot-spot residue on the substrate Cdk2 (Asp206) and confirm its interaction with hot-spot residues on Cdc25 using hot-spot swapping and double mutant cycles to derive interaction energies. Our experimentally validated model is consistent with previous studies of Cdk2 and its interaction partners and initiates the opportunity for drug discovery of inhibitors that target the remote binding sites of this protein-protein interaction.
Cdc25磷酸酶是真核细胞周期的关键激活因子，也是极具吸引力的抗癌靶点，因为它们的过表达与多种癌症相关。然而，针对这些磷酸酶的药物研发受到阻碍，原因是缺乏关于Cdc25如何与其天然蛋白质底物——细胞周期蛋白依赖性激酶相互作用的结构信息。在此，我们通过刚体对接方法预测了Cdc25B与其Cdk2-pTpY-CycA蛋白质底物的对接方向，并通过全尺度分子动力学模拟和最小化对对接模型进行优化。我们通过各种体外和体内技术对稳定的整体结构进行实验验证。具体而言，我们将我们的模型与Cdc25B底物捕获突变体的晶体结构进行比较。我们在体内鉴定并验证了Cdc25B上一个新的热点残基（Arg492），它在蛋白质底物识别中起核心作用。我们在底物Cdk2上鉴定出一个热点残基（Asp206），并使用热点交换和双突变循环来推导相互作用能，从而确认其与Cdc25上热点残基的相互作用。我们经实验验证的模型与之前关于Cdk2及其相互作用伙伴的研究一致，并为发现靶向这种蛋白质-蛋白质相互作用远程结合位点的抑制剂提供了机会。
