STRUCTURE - FUNCTION AND KINETICS OF AAA+ ATPASES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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，以拆除和重新组装膜或其他细胞器，复制DNA并穿越细胞分裂，修复受损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。我们的研究集中在AAA+ ATPAes的一个子集，细菌增强子结合蛋白（EBP），利用其ATP酶活性调节有害活动（疾病，作物损害）或有益活动（固氮，环境修复，氢或其他代谢产物生产）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ATP酶的第二种形式，它将ADP水解为AMP。发现AAA+ ATP酶的这种新活性是因为SAXS数据显示ADP在ATP酶的突变体中引起与ATP结合野生型蛋白引起的相同的构象变化。ATP酶与腺苷三磷酸双磷酸酶形式的色谱分离首次产生了这种特定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。

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.