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耦合蛋白 酶活性的动力学仍然很不完整。事实上，虽然我们对构象的理解 动力学介导的生物功能主要是基于对低复杂性，低 在分子量系统中，酶通常是寡聚的多结构域蛋白质，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。 如果没有一个全面的，原子分辨率的构象动力学介导的理解， 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，如变构和协同性，将继续落后。 在这里，我们将使用核磁共振结合其他生物物理和生物化学方法来揭示如何 辅因子/底物结合和构象动力学之间的复杂相互作用调节 高分子量酶是人体和细菌代谢所必需的。利益系统 在该提议中是细菌磷酸转移酶系统（PTS）的酶I（EI）和人RNA 脱甲基酶FTO和Alkbh 5。EI是一种128 kDa的二聚体酶，其活性取决于协同作用。 四个构象平衡的作用，导致一系列大的域内，域间， 由底物结合调节的亚基间结构重排。PTS是一个中央监管机构， 控制多种细胞功能的细菌代谢，包括毒力和生物膜形成， 磷酸化依赖性蛋白质-蛋白质相互作用。因此，在原子水平上理解EI活动将 阐明了蛋白质中长距离结构域间通讯的基本机制，并可能 提出了对抗细菌感染的新的治疗策略。本建议的第二部分 专注于能够催化N6-甲基腺苷（m6 A）氧化去甲基化的酶。 m6 A是真核生物mRNA中最丰富的修饰。动态调节m6 A修饰作用 在基因表达、细胞对外界刺激的反应、肿瘤形成、脂肪形成和细胞增殖中起重要作用。 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Alkbh 5。我们的研究结果将指导新的战略， 实现FTO和Alkbh 5的选择性抑制，以控制基因表达并对比FTO和Alkbh 5的进展。 癌总之，我的研究计划将阐明大规模构象之间的耦合 两种不同类型的高分子量多结构域酶的变化和功能， 对未来肥胖症和癌症的治疗以及新的抗生素靶点的见解。

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.