Collective Dynamics Differentiates Functional Divergence in Protein Evolution

Protein evolution is most commonly studied by analyzing related protein sequences and generating ancestral sequences through Bayesian and Maximum Likelihood methods, and/or by resurrecting ancestral proteins in the lab and performing ligand binding studies to determine function. Structural and dynamic evolution have largely been left out of molecular evolution studies. Here we incorporate both structure and dynamics to elucidate the molecular principles behind the divergence in the evolutionary path of the steroid receptor proteins. We determine the likely structure of three evolutionarily diverged ancestral steroid receptor proteins using the Zipping and Assembly Method with FRODA (ZAMF). Our predictions are within ∼2.7 Å all-atom RMSD of the respective crystal structures of the ancestral steroid receptors. Beyond static structure prediction, a particular feature of ZAMF is that it generates protein dynamics information. We investigate the differences in conformational dynamics of diverged proteins by obtaining the most collective motion through essential dynamics. Strikingly, our analysis shows that evolutionarily diverged proteins of the same family do not share the same dynamic subspace, while those sharing the same function are simultaneously clustered together and distant from those, that have functionally diverged. Dynamic analysis also enables those mutations that most affect dynamics to be identified. It correctly predicts all mutations (functional and permissive) necessary to evolve new function and ∼60% of permissive mutations necessary to recover ancestral function.蛋白质进化研究最常用的手段包括分析同源蛋白质序列，通过贝叶斯（Bayesian）和最大似然（Maximum Likelihood）方法重建祖先序列，或是在实验室中复活祖先蛋白质并开展配体结合实验以确定其功能。但分子进化研究在很大程度上忽略了结构与动态层面的演化过程。本研究同时结合结构与动态特征，以阐明类固醇受体蛋白进化路径分化背后的分子原理。我们借助结合FRODA的折叠组装法（Zipping and Assembly Method with FRODA, ZAMF），预测了3种进化分化的祖先类固醇受体蛋白的潜在结构。我们的预测结果与祖先类固醇受体对应晶体结构的全原子均方根偏差（RMSD）仅约2.7 Å。除静态结构预测外，ZAMF的一大特色是可生成蛋白质动态信息。本研究通过本质动力学（essential dynamics）获取蛋白质的集体运动模式，以此分析分化后蛋白质构象动态的差异。值得注意的是，本研究分析发现，同一家族中进化分化的蛋白质并不共享相同的动态子空间；而功能一致的蛋白质则会聚集在一起，与功能分化的蛋白质相互分离。动态分析还可用于筛选对蛋白质动态影响最大的突变位点。该方法可准确预测获得新功能所需的全部突变（包括功能突变与允许突变），以及恢复祖先功能所需的约60%的允许突变。