Atomic-level characterization of self-regulatory mechanisms in large multidomain enzymes

项目总结/摘要 酶是非凡的纳米机器，在细胞代谢中发挥着无数重要功能。 通过辅因子/底物结合调节酶的结构和灵活性提供了一个重要的来源， 调节酶的功能，但我们的理解的基本机制，协调蛋白质 运动促进酶活性在很大程度上仍不完全。事实上，虽然有几项研究已经出现在 在过去的二十年里，我们描述了构象动力学如何介导小蛋白质的生物学功能， 了解多重构象平衡之间的耦合如何决定大分子的活性 多域系统继续滞后。本提案的总体目标是使用和开发综合 将NMR与互补的生物物理和生物化学工具相结合的方法，以揭示 辅因子/底物结合后的局部紊乱影响协同运动，并调节高水平的 分子量的酶是人体和细菌代谢所必需的。这种工具组合 对蛋白质运动的敏感性带来了高分子量酶功能的新的详细图像。的 在本发明中表征的酶是细菌磷酸转移酶系统（PTS）的酶I（EI）， 和核酸脱甲基酶的AlkB家族--这些不同类别的酶一起显示了 通过这种工具的组合，可以探索局域无序和协同磁畴运动之间的关系 并证明这些机制在不同的酶类别中是多么重要。特别是，EI 酶的活性取决于四种构象平衡的协同作用，这导致一系列的 由底物结合调节的大的结构域内、结构域间和亚基间结构重排。 因此，我们在原子水平上揭示EI功能的努力将揭示局部无序的调节如何介导 长距离域间通信，并最终调节这种必需的细菌酶的活性。 AlkB双加氧酶是一种灵活的酶，已知其内部动力学受到调节 在基底结合时。我们的研究将使apo和holo AlkB酶中的构象紊乱可视化， 前所未有的原子分辨率的细节，并将揭示如何在活性位点的残余无序决定 底物选择性此外，我们还将研究AlkB蛋白与其受体形成的许多复合物。 抑制剂的我们希望这些结果表明新的策略，基于构象的选择性扰动， 疾病，开发具有亚家族选择性的AlkB抑制剂。总之，我的研究计划将阐明 两类不同高分子大尺度构象变化与功能的耦合 重量多结构域酶，为未来的肥胖和癌症治疗提供新的见解， 抗生素靶点此外，这些努力推动了生物物理工具应用的前沿， 原子分辨复杂酶中无序与功能协同运动的关系 提供了一个模板转移到未知的多域系统的机制调查。

PROJECT SUMMARY/ABSTRACT Enzymes are remarkable nanomachines that play a myriad of essential functions in cellular metabolism. Modulation of enzyme structure and flexibility by cofactor/substrate binding provides an important source of regulation of enzyme function, yet our understanding of the fundamental mechanisms by which concerted protein motion facilitate enzymatic activity is still largely incomplete. Indeed, while several studies have appeared in the past two decades describing how conformational dynamics mediate the biological function of small proteins, our understanding of how the coupling among multiple conformational equilibria determines the activity of large multidomain systems continues to lag. The overall goal of this proposal is using and developing integrated approaches combining NMR with complementary biophysical and biochemical tools to reveal how modulation of local disorder upon cofactor/substrate binding affects concerted motions and regulates the activity of high molecular weight enzymes that are essential for human and bacterial metabolism. This combination of tools sensitive to protein motion brings a newly detailed picture of high molecular weight enzyme function. The enzymes characterized in this proposal are Enzyme I (EI) of the bacterial phosphotransferase system (PTS), and the AlkB family of nucleic acid demethylases – together these distinct classes of enzymes show how the relationship between local disorder and concerted domain motions can be probed by this combination of tools and demonstrate how essential these mechanisms are across diverse enzyme classes. In particular, the EI enzymatic activity depends upon the synergistic action of four conformational equilibria that results in a series of large intradomain, interdomain, and intersubunit structural rearrangements modulated by substrate binding. Therefore, our efforts to uncover EI function at atomic level will reveal how modulation of local disorder mediates long-range interdomain communication and, ultimately, regulates the activity of this essential bacterial enzyme. The AlkB dioxygenases are flexible enzymes that are known to undergo modulation of their internal dynamics upon substrate binding. Our studies will visualize conformational disorder in apo and holo AlkB enzymes with unprecedented atomic-resolution details, and will reveal how residual disorder at the active site determines substrate selectivity. In addition, we will investigate a number of complexes formed by AlkB proteins with their inhibitors. We expect these results to indicate new strategies, based on selective perturbation of conformational disorder, to develop AlkB inhibitors with subfamily selectivity. In summary, my research program will elucidate the coupling between large scale conformational changes and function in two distinct classes of high molecular weight multidomain enzymes, providing new insights for future therapies for obesity and cancer as well as novel antibiotic targets. Moreover, these efforts pushing the frontier of the application of biophysical tools to study with atomic resolution the relationship between disorder and functional concerted motions in complex enzymes provide a template transferrable to mechanistic investigations of uncharted multi-domain systems.