Effect of the Flexible Regions of the Oncoprotein Mouse Double Minute X on Inhibitor Binding Affinity
Effect of the Flexible Regions of the Oncoprotein Mouse Double Minute X on Inhibitor Binding Affinity
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癌蛋白小鼠双分钟 X 的柔性区域对抑制剂结合亲和力的影响
DOI：
10.1021/acs.biochem.7b00903
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发表时间：
2017
期刊：
影响因子：
2.9
通讯作者：
Su Zhengding
中科院分区：
文献类型：
--
作者：
Qin Lingyun;Liu Huili;Chen Rong;Zhou Jingjing;Cheng Xiyao;Chen Yao;Huang Yongqi;Su Zhengding
癌蛋白MdmX（小鼠双微体X）在其氨基酸序列和三维构象方面与Mdm 2（小鼠双微体2）高度同源，但Mdm 2抑制剂对MdmX表现出非常弱的亲和力，为探索蛋白质构象如何区分和改变抑制剂结合提供了极好的模型。蛋白质固有的构象灵活性在决定和预测蛋白质的结合性质以及抑制剂的设计中起着关键作用。虽然分子动力学模拟方法使我们能够理解蛋白质-配体相互作用，但柔性结合口袋如何适应抑制剂的机制在实验上探索较少。在这项工作中，我们研究了如何使用蛋白质工程的MdmX（N-MdmX）的N-末端结构域的内在灵活的区域影响的Mdm 2抑制剂nutlin-3a的亲和力。在异源核Overhauser效应测量的指导下，我们确定了影响N-MdmX上配体结合口袋周围抑制剂结合亲和力的灵活区域。二硫键工程突变体N-MdmXC 25-C110/C76-C88，其结合了两个斯台普斯以刚性化配体结合口袋，允许对nutlin-3a的亲和力高于野生型N-MdmX（Kd = 0.48 vsKd = 20.3 μM）。因此，该突变体不仅为MdmX抑制剂的筛选和设计提供了有效的蛋白质模型，而且为增强具有明显柔性区域的配体的药效团的分子间相互作用提供了有价值的线索。此外，我们的研究结果揭示了N-MdmX的变构配体结合机制，其中配体最初与紧凑的核心相互作用，随后增强与固有柔性区域的分子间相互作用。这种策略也应该适用于许多其他蛋白质靶点，以加速药物发现。
The oncoprotein MdmX (mouse double minute X) is highly homologous to Mdm2 (mouse double minute 2) in terms of their amino acid sequences and three-dimensional conformations, but Mdm2 inhibitors exhibit very weak affinity for MdmX, providing an excellent model for exploring how protein conformation distinguishes and alters inhibitor binding. The intrinsic conformation flexibility of proteins plays pivotal roles in determining and predicting the binding properties and the design of inhibitors. Although the molecular dynamics simulation approach enables us to understand protein–ligand interactions, the mechanism underlying how a flexible binding pocket adapts an inhibitor has been less explored experimentally. In this work, we have investigated how the intrinsic flexible regions of the N-terminal domain of MdmX (N-MdmX) affect the affinity of the Mdm2 inhibitor nutlin-3a using protein engineering. Guided by heteronuclear nuclear Overhauser effect measurements, we identified the flexible regions that affect inhibitor binding affinity around the ligand-binding pocket on N-MdmX. A disulfide engineering mutant, N-MdmXC25–C110/C76–C88, which incorporated two staples to rigidify the ligand-binding pocket, allowed an affinity for nutlin-3a higher than that of wild-type N-MdmX (Kd∼ 0.48 vsKd∼ 20.3 μM). Therefore, this mutant provides not only an effective protein model for screening and designing of MdmX inhibitors but also a valuable clue for enhancing the intermolecular interactions of the pharmacophores of a ligand with pronounced flexible regions. In addition, our results revealed an allosteric ligand-binding mechanism of N-MdmX in which the ligand initially interacts with a compact core, followed by augmenting intermolecular interactions with intrinsic flexible regions. This strategy should also be applicable to many other protein targets to accelerate drug discovery.