STRUCTURE - FUNCTION AND KINETICS OF AAA+ ATPASES

AAA 腺苷酸酶的结构 - 功能和动力学

• 批准号: 7722747
• 负责人: B TRACY NIXON
• 金额: $ 1.26万
• 依托单位: ILLINOIS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722747
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Apyrase; Binding; Cell division; Cells; Computer Retrieval of Information on Scientific Projects Database; DNA; Data; Defect; Disease; Funding; Gene Expression; Genetic Transcription; Grant; Human; Hydrogen; Hydrolysis; Institution; Kinetics; Mechanics; Membrane; Molecular Machines; Nitrogen Fixation; Organelles; Outcome; Production; Proteins; Research; Research Personnel; Resources; Source; Structure; Time; United States National Institutes of Health; Work; enhancer binding protein; mutant; novel; pathogen; remediation; repaired

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Why do defects in molecular machines (AAA+ ATPases) cause disease? These protein machines convert ATP hydrolysis into mechanical work. Both human cells and disease causing pathogens use this work to physically manipulate proteins or DNA to dismantle and reassemble membranes or other organelles, to replicate DNA and traverse cell division, to repair damaged proteins, or to regulate gene expression. We do not know how these molecular machines convert ATP hydrolysis into mechanical work. Our research focuses on one subset of AAA+ ATPAses, the bacterial-enhancer-binding proteins (EBPs) which use their ATPase activities to regulate transcription of genes needed for harmful activites (diseases, crop damage) or helpful ones (nitrogen fixation, environmental remediation, hydrogen or other metabolite production). In a prior project, we established two mechanisms for regulating the EBP ATPases, and began defining structural changes occuring in their catalytic cycle. The current project completes that objective, also extending it to address the underlying mechanism via structure function studies of mutant forms of ATPase. An unexpected outcome was the discovery of a second form of the ATPase that hydrolyzes ADP to AMP. This novel activity for AAA+ ATPases was discovered because SAXS data showed ADP causing the same conformational changes in a mutant of the ATPase that are caused by ATP binding to the wild type protein. Chromatographic separation of the ATPase from the apyrase form yielded for the first time a crystal structure of the ATP-bound form of this particular AAA+ ATPase, allowing us to further interpret prior SAXS data.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 为什么分子机器（AAA+ ATP酶）的缺陷会导致疾病？这些蛋白质机器将 ATP 水解转化为机械功。人类细胞和致病病原体都利用这项工作来物理操纵蛋白质或 DNA，以拆卸和重新组装膜或其他细胞器、复制 DNA 和遍历细胞分裂、修复受损的蛋白质或调节基因表达。我们不知道这些分子机器如何将 ATP 水解转化为机械功。我们的研究重点是 AAA+ ATPA 酶的一个子集，即细菌增强子结合蛋白 (EBP)，它们利用 ATP 酶活性来调节有害活动（疾病、作物损害）或有益活动（固氮、环境修复、氢气或其他代谢物生产）所需的基因转录。 在之前的项目中，我们建立了两种调节 EBP ATP 酶的机制，并开始定义其催化循环中发生的结构变化。当前的项目完成了这一目标，并将其扩展到通过 ATP 酶突变形式的结构功能研究来解决潜在机制。一个意想不到的结果是发现了第二种形式的 ATP 酶，可将 ADP 水解为 AMP。 AAA+ ATP 酶的这种新活性被发现是因为 SAXS 数据显示 ADP 在 ATP 酶突变体中引起与 ATP 与野生型蛋白结合引起的相同构象变化。将 ATP 酶与腺苷三磷酸双磷酸酶形式进行色谱分离，首次获得了这种特定 AAA+ ATP 酶的 ATP 结合形式的晶体结构，使我们能够进一步解释先前的 SAXS 数据。