Department of Pharmaceutical Sciences , University of Maryland School of Pharmacy , Baltimore , Maryland 21201 , United States.
J Am Chem Soc. 2019 Sep 25;141(38):15092-15101. doi: 10.1021/jacs.9b06064. Epub 2019 Sep 13.
Protein kinases are important cellular signaling molecules involved in cancer and a multitude of other diseases. It is well-known that inactive kinases display a remarkable conformational plasticity; however, the molecular mechanisms remain poorly understood. Conformational heterogeneity presents an opportunity but also a challenge in kinase drug discovery. The ability to predictively model various conformational states could accelerate selective inhibitor design. Here we performed a proton-coupled molecular dynamics study to explore the conformational landscape of a c-Src kinase. Starting from a completely inactive structure, the simulations captured all major types of conformational states without the use of a target structure, mutation, or bias. The simulations allowed us to test the experimental hypotheses regarding the mechanism of DFG flip, its coupling to the αC-helix movement, and the formation of regulatory spine. Perhaps the most significant finding is how key titratable residues, such as DFG-Asp, αC-Glu, and HRD-Asp, change protonation states dependent on the DFG, αC, and activation loop conformations. Our data offer direct evidence to support a long-standing hypothesis that protonation of Asp favors the DFG-out state and explain why DFG flip is also possible in simulations with deprotonated Asp. The simulations also revealed intermediate states, among which a unique DFG-out/α-C state formed as DFG-Asp is moved into a back pocket forming a salt bridge with catalytic Lys, which can be tested in selective inhibitor design. Our finding of how proton coupling enables the remarkable conformational plasticity may shift the paradigm of computational studies of kinases which assume fixed protonation states. Understanding proton-coupled conformational dynamics may hold a key to further innovation in kinase drug discovery.
蛋白激酶是参与癌症和多种其他疾病的重要细胞信号分子。众所周知，非活性激酶表现出显著的构象灵活性；然而，其分子机制仍知之甚少。构象异质性为激酶药物发现带来了机遇，但也带来了挑战。能够预测各种构象状态的能力可以加速选择性抑制剂的设计。在这里，我们进行了质子偶联分子动力学研究，以探索 c-Src 激酶的构象景观。从一个完全非活性的结构开始，模拟捕获了所有主要类型的构象状态，而无需使用目标结构、突变或偏见。模拟使我们能够测试关于 DFG 翻转机制、与αC-螺旋运动的耦合以及调节脊柱形成的实验假设。也许最显著的发现是，关键的可滴定残基（如 DFG-Asp、αC-Glu 和 HRD-Asp）如何根据 DFG、αC 和激活环构象改变质子化状态。我们的数据提供了直接证据，支持了一个长期存在的假设，即 Asp 的质子化有利于 DFG-out 状态，并解释了为什么在去质子化 Asp 的模拟中也可能发生 DFG 翻转。模拟还揭示了中间状态，其中形成了一种独特的 DFG-out/α-C 状态，其中 DFG-Asp 被移动到后口袋中，与催化 Lys 形成盐桥，这可以在选择性抑制剂设计中进行测试。我们发现质子偶联如何使显著的构象灵活性成为可能，这可能会改变激酶计算研究的范例，这些研究假设质子化状态是固定的。理解质子偶联的构象动力学可能是激酶药物发现进一步创新的关键。
