Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy
Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy
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单分子光谱揭示 Aurora A 激酶激活环的动态平衡
DOI：
10.1002/ange.201704654
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发表时间：
2017
影响因子：
--
通讯作者：
Gilburt J
中科院分区：
文献类型：
--
作者：
Gilburt J
蛋白激酶激活环（T环）的构象是酶活性的基础，并影响小分子抑制剂的结合。通过使用单分子荧光光谱，我们已经确定磷酸化的Aurora A激酶处于DFG-in-样活性T-环构象和DFG-out-样非活性构象之间的动态平衡，并测量了相互转化的速率常数。 添加Aurora A活化蛋白TPX 2使平衡向活性T环构象移动，而添加抑制剂MLN 8054和CD 532有利于无活性T环。 我们发现Aurora A同时结合TPX 2和MLN 8054，并为激酶构象行为提供了一个新的模型。 我们的方法将使构象特异性效应能够整合到整个激酶组的抑制剂发现中，并且我们概述了基于结构的药物发现的一些直接后果。
The conformation of the activation loop (T‐loop) of protein kinases underlies enzymatic activity and influences the binding of small‐molecule inhibitors. By using single‐molecule fluorescence spectroscopy, we have determined that phosphorylated Aurora A kinase is in dynamic equilibrium between a DFG‐in‐like active T‐loop conformation and a DFG‐out‐like inactive conformation, and have measured the rate constants of interconversion. Addition of the Aurora A activating protein TPX2 shifts the equilibrium towards an active T‐loop conformation whereas addition of the inhibitors MLN8054 and CD532 favors an inactive T‐loop. We show that Aurora A binds TPX2 and MLN8054 simultaneously and provide a new model for kinase conformational behavior. Our approach will enable conformation‐specific effects to be integrated into inhibitor discovery across the kinome, and we outline some immediate consequences for structure‐based drug discovery.