Evolution has altered the free energy landscapes of protein kinases to introduce different regulatory switches and modify their catalytic functions. In this work, we demonstrate how cyclin dependency has emerged in cyclin-dependent kinases (CDKs) by reconstructing their closest experimentally characterized cyclin-independent ancestor, CMGI, using molecular dynamics simulations. Four hypotheses are formulated to describe why CDKs require an additional regulatory switch, i.e. cyclin binding to adopt an active state. Each hypothesis is tested using all-atom molecular dynamics simulations of CDK2 and the ancestor. In both systems, the K33–E51 hydrogen bond and the alignment of regulatory-spine residues have similar stabilities. However, auto-inhibition due to a helical turn in the A-loop is observed to be less favorable in the ancestor. Unlike the ancestor, the aspartate of the DFG motif does not form a bidentate bond with a Mg²⁺ ion in CDK2. These results explain the experimental observation of cyclin independency of the ancestor. Our findings provide a mechanistic rationale for how evolution has added a new regulatory switch to CDKs to tightly regulate the signalling pathways. This approach is directly applicable to other proteins to study the emergence of different types of regulatory mechanisms.

中文翻译：

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进化如何设计蛋白质的功能自由能态？以细胞周期蛋白依赖性激酶家族调控出现为例

进化改变了蛋白激酶的自由能态，以引入不同的调节开关并改变其催化功能。在这项工作中，我们通过使用分子动力学模拟重建它们最接近的实验表征的细胞周期独立性祖先CMGI，来证明细胞周期蛋白依赖性激酶（CDK）中如何出现细胞周期蛋白依赖性。提出了四个假设来描述CDK为什么需要额外的监管转换，即细胞周期蛋白结合采取活跃状态。每个假设都使用CDK2和祖先的全原子分子动力学模拟进行测试。在这两个系统中，K33–E51氢键和调节性脊柱残基的排列都具有相似的稳定性。但是，在祖先中观察到由于A环中的螺旋形转弯而引起的自抑制作用较差。与祖先不同，DFG基序的天冬氨酸不会与Mg ²⁺形成双齿键CDK2中的离子。这些结果解释了祖先细胞周期蛋白独立性的实验观察。我们的发现为进化如何为CDK添加了新的调控开关以紧密调控信号传导途径提供了机械原理。该方法直接适用于其他蛋白质，以研究不同类型调节机制的出现。