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

大型多域酶自我调节机制的原子水平表征

• 批准号: 10408689
• 负责人: Vincenzo Venditti
• 金额: $ 36.78万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10408689
• 关键词: Antibiotics; Bacterial Infections; Binding; Biochemical; Biological Process; Biology; Biophysics; Cell physiology; Communication; Complex; Coupling; Development; Enzymes; Future; Gene Expression; Human; Malignant Neoplasms; Mediating; Messenger RNA; Metabolism; Microbial Biofilms; Modification; Molecular Conformation; Molecular Weight; Phosphorylation; Phosphotransferases; Play; Protein Dynamics; Proteins; RNA; Regulation; Research; Resolution; Role; Series; Source; Stimulus; System; Virulence; bacterial metabolism; base; cofactor; combat; demethylation; dimer; enzyme structure; flexibility; human disease; insight; interest; lipid biosynthesis; nanomachine; novel; novel strategies; novel therapeutic intervention; obesity treatment; programs; protein protein interaction; response; small molecule; tumor progression; tumorigenesis

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.

项目摘要/摘要 酶是显着的纳米机器，在细胞代谢中起着无数的基本功能。 辅助因子/底物结合对酶结构和柔韧性的调节提供了重要的来源 调节酶功能，但是我们对基本机制耦合蛋白的理解 酶活性的动力学基本上是不完整的。确实，尽管我们对如何构象的欣赏 动力学介导生物学功能主要基于低复杂性，低 - 分子量体重系统，酶通常是寡聚，多域蛋白的生物学功能 取决于内域，域间和亚基间构象平衡之间的复杂耦合。 没有对构象动力学介导的自我介导的全面，原子分辨率的理解 高复杂性，高分子重量酶的调节机制，我们理解和 生物学中的普遍现象（例如变构和合作）将继续落后。 在这里，我们将使用NMR与其他生物物理和生化方法结合在一起，以揭示如何揭示如何 辅因子/底物结合和构象动力学之间的复杂相互作用调节 对人和细菌代谢必不可少的高分子量酶。感兴趣的系统 在该提案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ALKBH5。 EI是一种128 kDa二聚体酶，其活性取决于协同作用 四个构象平衡的作用，导致一系列大型内域，室内和 底物结合调节的亚基间结构重排。 PTS是 通过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，了解原子水平的EI活动将 阐明管理蛋白质长距离交流的基本机制，可能 提出新的治疗策略来打击细菌感染。本提案的第二部分 侧重于能够催化N6-甲基二糖苷（M6A）的氧化脱甲基化的酶。 M6A是真核mRNA中最丰富的修饰。 M6A修饰戏剧的动态调节 在基因表达，对外部刺激的细胞反应，肿瘤发生，成生成和中的重要作用 其他人类疾病的发展。我们将研究调节功能的机制 具有原子分辨率的人RNA去甲基酶FTO和ALKBH5。我们的结果将指导新策略 实现对FTO和ALKBH5的选择性抑制以控制基因表达和对比的进展 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 两种不同类别的高分子重量多域酶的变化和功能，提供了新的 对肥胖和癌症的未来疗法以及新型抗生素靶标的见解。