NUCLEOTIDE DEPENDENT CONFORMATIONAL CHANGES IN S54-DEPENDENT AAA+ATPASES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。为什么分子机器（AAA+ atp酶）缺陷会导致疾病？这些蛋白质机器将ATP水解转化为机械功。很明显，人类细胞和致病病原体都利用这项工作来物理地操纵蛋白质或DNA来拆除和重新组装膜或其他细胞器，复制DNA和穿越细胞分裂，修复受损的蛋白质，或调节基因表达。但是，我们不知道这些分子机器是如何将ATP水解转化为机械功的。定义这些机制的一个主要障碍是我们无法探测与控制这些ATP酶有关的详细构象变化，因为它们通过ATP结合，水解和产物释放。我们的项目专注于定义结构变化，以调节554依赖的AAA+ ATP酶的组装，并伴随着ATP水解循环。这些激活剂重塑细菌RNA聚合酶的554形式，使其能够在细菌中启动转录。先前从小角度散射数据得出的溶液结构（发表于2005年）表明，在一种情况下，调节结构域采用两种同质二聚体形式，交替抑制或抑制相邻atp酶结构域的马达组装；在另一种情况下，发现调节和atp酶结构域合作稳定组装的马达。2005年收集的类似数据揭示了a)与核苷酸状态耦合的构象变化，并介导了与靶蛋白s54的两种不同的相互作用；b)其中一种复合物的结构。后一种结果正在提交出版。

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. It is clear that 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. But, we do not know how these molecular machines convert ATP hydrolysis into mechanical work. A major impediment to defining these mechanisms has been our inability to probe detailed conformational changes that are related to controlling these ATPases as they step through ATP binding, hydrolysis and product release. Our project focused on defining structural changes that occur to regulate assembly of s54-dependent AAA+ ATPases, and that accompany the ATP hydrolysis cycle. These activators remodel the s54-form of bacterial RNA polymerase to enable it to initiate transcription in bacteria. Previous solution structures derived from small angle scattering data (published in 2005) revealed that in one case a regulatory domain adopts two homo-dimeric forms, alternately repressing or derepressing motor assembly by adjacent ATPase domains; in another case, regulatory and ATPase domains were found to cooperate to stabilize the assembled motor. Similar data collected in 2005 revealed a) conformational changes that are coupled with nucleotide status and that mediate two distinct interactions with the target protein, s54, and b) structure of one of those complexes. These latter results are being submitted for publication.