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

项目摘要/摘要 酶是非凡的纳米机器，在细胞新陈代谢中发挥着无数的基本功能。 辅因子/底物结合的酶结构和灵活性的调节提供了一个重要的来源 酶功能的调节，但我们对偶联蛋白质的基本机制的理解 酶活性的动力学仍在很大程度上不完整。事实上，虽然我们对构象的欣赏 动力学中介生物功能的主要基础是低复杂性、低成本和低成本的结构研究。 分子量系统，酶是典型的寡聚、多结构域蛋白质，其生物学功能 依赖于结构域内、结构域间和亚基间构象平衡之间的复杂耦合。 没有对构象动力学的全面的、原子分辨率的理解--介导的、自我的 高复杂性、高分子量酶的调节机制，我们理解和 利用生物学中普遍存在的现象，如变构和协作性，将继续滞后。 在这里，我们将使用核磁共振结合其他生物物理和生化方法来揭示 辅因子/底物结合和构象动力学之间的复杂相互作用调节酶的活性 对人类和细菌新陈代谢必不可少的高分子量酶。有关利益的制度 在这项建议中是细菌磷酸转移酶系统(PTS)的酶I(EI)，以及人类的RNA 去甲基化酶FTO和Alkbh5。EI是一种128 kDa的二聚体酶，其活性依赖于协同作用 四种构象平衡的作用，导致一系列大的结构域内、结构域间和 底物结合调节的亚基间结构重排。临时秘书处是一个中央监管机构， 控制多种细胞功能的细菌代谢，包括毒力和生物膜的形成，通过 依赖于磷酸化的蛋白质-蛋白质相互作用。因此，在原子水平上理解EI活动将 阐明了管理蛋白质中远程域间通信的基本机制，并可能 提出新的治疗策略来对抗细菌感染。本提案的第二部分 重点介绍能够催化N6-甲基腺苷(M6A)氧化去甲基化的酶。 M6A是真核基因中含量最丰富的修饰。M6A修饰剧的动态调整 在基因表达、细胞对外界刺激的反应、肿瘤发生、脂肪形成和 其他人类疾病的发展。我们将研究调节细胞功能的机制 人RNA去甲基化酶FTO和Alkbh5具有原子分辨率。我们的结果将指导新的战略 实现对FTO和Alkbh5的选择性抑制以控制基因表达和对比进展 癌症。总而言之，我的研究计划将阐明大规模构象之间的耦合 两类不同类型的高分子量多结构域酶的变化和功能，提供了新的 对未来治疗肥胖症和癌症的见解以及新的抗生素靶点。

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 coupling protein dynamics to enzymatic activity is still largely incomplete. Indeed, while our appreciation of how conformational dynamics mediate biological function is predominantly based on structural studies on low-complexity, low- molecular weight systems, enzymes are typically oligomeric, multidomain proteins whose biological function depends on an intricate coupling among intradomain, interdomain, and intersubunit conformational equilibria. Without a comprehensive, atomic-resolution understanding of conformational dynamics-mediated, self- regulatory mechanisms in high-complexity, high-molecular weight enzymes, our ability to understand and exploit ubiquitous phenomena in biology, such as allosterism and cooperativity, will continue to lag. Here, we will use NMR combined with other biophysical and biochemical approaches to reveal how the complex interplay between cofactor/substrate binding and conformational dynamics regulates the activity of high molecular weight enzymes that are essential for human and bacterial metabolism. The systems of interest in this proposal are Enzyme I (EI) of the bacterial phosphotransferase system (PTS), and the human RNA demethylases FTO and Alkbh5. EI is a 128 kDa dimeric enzyme whose activity depends on the synergistic action of four conformational equilibria that results in a series of large intradomain, interdomain, and intersubunit structural rearrangements modulated by substrate binding. The PTS is a central regulator of bacterial metabolism that controls multiple cellular functions, including virulence and biofilm formation, through phosphorylation-dependent protein-protein interactions. Therefore, understanding EI activity at atomic level will illuminate the fundamental mechanisms governing long-range interdomain communication in proteins, and may suggest new therapeutic strategies to combat bacterial infections. The second part of the present proposal focuses on enzymes that are capable of catalyzing oxidative demethylation of the N6-methyladenosine (m6A). m6A is the most abundant modification in eukaryotic mRNA. Dynamic regulation of the m6A modification plays an important role in gene expression, cellular response to external stimuli, oncogenesis, adipogenesis and in development of other human diseases. We will investigate the mechanisms that regulate the function of the human RNA demethylases FTO and Alkbh5 with atomic resolution. Our results will guide new strategies to achieve selective inhibition of FTO and Alkbh5 to control gene expression and to contrast progression of cancer. 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.