Mechanism of an Acid Activated Chaperone

描述（由申请人提供）：分子伴侣HdeA是一种应激特异性伴侣，在低ph条件下迅速激活，以保护蛋白质免受广泛的酸诱导的蛋白质聚集。这种应激特异性激活HdeA对于大肠杆菌等致病菌在胃的低pH抗菌环境中生存是必不可少的。HdeA在蛋白质水平上感知应激条件，并通过自身的快速展开作出反应，使HdeA成为一类新的伴侣蛋白的成员，这种蛋白需要失去结构才能获得活性。最近也报道了其他atp无关的伴侣蛋白通过部分展开进行类似的应激特异性激活，这表明这种机制可能代表了伴侣蛋白和内在无序蛋白领域的新范式。我们现在将使用结构、生化和突变工具的组合来阐明i) HdeA是如何被激活的，ii)在应激条件下允许HdeA与各种不同的客户蛋白结合的特征，以及iii)在返回非应激条件时支持其客户蛋白重折叠的机制。这些研究将揭示两个非常普遍的未解问题的答案：蛋白质如何结合多个不相关的客户蛋白，以及伴侣蛋白如何与客户蛋白相互作用以促进其再折叠。因此这项工作将

DESCRIPTION (provided by applicant): The molecular chaperone HdeA is a stress-specific chaperone that is rapidly activated by low pH-conditions to protect proteins against widespread acid-induced protein aggregation. This stress specific activation of HdeA is essential for pathogenic bacteria such as E. coli to survive the low pH antimicrobial environment of the stomach. HdeA senses the stress conditions at the protein level and responds to it with its own very rapid unfolding, making HdeA a member of a new class of chaperones, which need to lose structure to gain activity. Similar stress-specific activation by partial unfolding has been recenty reported for other ATP-independent chaperones as well, suggesting that this mechanism may represent a new paradigm in the field of chaperones and intrinsically disordered proteins. We will now use a combination of structural, biochemical and mutational tools to elucidate i) how HdeA becomes activated, ii) the features that allow HdeA to bind to a wide variety of different client proteins under stress conditions and iii) the mechanism by which it supports refolding of its client proteins upon return to non-stress conditions. These studies will reveal the answers to two very general unanswered questions: how proteins bind multiple unrelated client proteins, and how chaperones interact with client proteins to facilitate their refolding. Thus this work will simultaneously address fundamental and as yet unresolved questions in two major fields, the field of molecular recognition and the field of chaperone action. HdeA is ideally suited for these studies. It is a small 10 kDa intrinsically disordered chaperone, which is highly amendable to structural analysis by NMR. It forms very stable interactions with a number of unrelated small client proteins with known NMR structures, and HdeA supports client refolding in the absence of co- chaperones or energy sources. Moreover, client binding and client release from HdeA is easily and precisely controlled by simple pH shifts. We will perform detailed NMR residue-level studies of dynamics and disorder in HdeA as a function of pH to assess how pH-changes are used for the controlled unfolding and activation of a chaperone. We will conduct residue-level NMR studies on HdeA and client proteins to simultaneously determine how HdeA impacts its client proteins and how client proteins affect HdeA. We will use in vivo folding biosensors to directly select for HdeA mutants with altered flexibility to assess the role that flexibility playsin chaperone function, and we will conduct refolding studies with select folding-mutants of HdeA's client protein immunity protein 7 to determine how HdeA promotes client refolding. Together, these studies will enable us to characterize the molecular mechanism that underlies HdeA's chaperone activity and test the hypothesis that flexibility is required for HdeA's molecular recognition function. With these studies, we now have an unprecedented opportunity to understand, in molecular detail, both the mechanism of chaperone action and the role of disorder in protein function and molecular recognition.