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Abstract：
Modulating the transitions between active and inactive conformations of protein kinases is the primary means of regulating their catalytic activity, achieved by phosphorylation of the activation loop (A-loop). To elucidate the mechanism of this conformational activation, we applied the string method to determine the conformational transition path of insulin receptor kinase between the active and inactive conformations and the corresponding free-energy profiles with and without A-loop phosphorylation. The conformational change was found to proceed in three sequential steps: first, the flipping of the DFG motif of the active site; second, rotation of the A-loop; finally, the inward movement of the αC helix. The main energetic bottleneck corresponds to the conformational change in the A-loop, while changes in the DFG motif and αC helix occur before and after A-loop conformational change, respectively. In accordance with this, two intermediate states are identified, the first state just after the DFG flipping and the second state after the A-loop rotation. These intermediates exhibit structural features characteristic of the corresponding inactive and active conformations of other protein kinases. To understand the impact of A-loop phosphorylation on kinase conformation, the free energies of A-loop phosphorylation were determined at several states along the conformational transition path using the free-energy perturbation simulations. The calculated free energies reveal that while the unphosphorylated kinase interconverts between the inactive and active conformations, A-loop phosphorylation restricts access to the inactive conformation, thereby increasing the active conformation population. Overall, this study suggests a consensus mechanism of conformational activation between different protein kinases.
摘要：
调节蛋白激酶的活性和非活性构象之间的转换是调节其催化活性的主要手段。通过激活环(A环)的磷酸化实现。为了阐明这种构象激活的机制，我们应用字符串方法来确定胰岛素受体激酶在活性和非活性构象之间的构象转换路径以及具有和不具有A环磷酸化的相应自由能谱.发现构象变化按三个顺序进行：首先，活性位点的DFG基序的翻转；第二，A循环的旋转;最后,αC螺旋的向内运动。主要的能量瓶颈对应于A环的构象变化，而DFG基序和αC螺旋的变化发生在A环构象变化之前和之后，分别。据此,识别两个中间状态，在DFG翻转之后的第一状态和在A循环旋转之后的第二状态。这些中间体表现出其他蛋白激酶的相应无活性和活性构象的结构特征。了解A环磷酸化对激酶构象的影响,使用自由能扰动模拟确定了沿构象转变路径的几种状态下A环磷酸化的自由能。计算的自由能表明，虽然未磷酸化的激酶在非活性和活性构象之间相互转换，A环磷酸化限制了进入非活性构象，从而增加了活性构象群体。总的来说,这项研究提示了不同蛋白激酶之间构象激活的共识机制。
