Mechanism of an Acid Activated Chaperone

描述（由申请方提供）：分子伴侣HdeA是一种应激特异性伴侣，可在低pH条件下快速激活，以保护蛋白质免受广泛的酸诱导蛋白质聚集的影响。HdeA的这种应激特异性激活对于致病菌如大肠杆菌是必需的。大肠杆菌存活的胃的低pH值的抗菌环境。HdeA在蛋白质水平上感知应激条件，并以其自身非常快速的解折叠对其做出反应，使HdeA成为一类新的伴侣蛋白的成员，其需要失去结构以获得活性。类似的应力特异性激活部分解折叠最近已被报道为其他ATP-独立的伴侣，以及，这表明这种机制可能代表了伴侣和内在无序蛋白质领域的一个新的范例。我们现在将使用结构，生物化学和突变工具的组合来阐明i）HdeA如何被激活，ii）允许HdeA在应激条件下与各种不同的客户蛋白结合的特征，以及iii）在返回到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回答的问题的答案：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重折叠。这项工作将 同时解决基本的和尚未解决的问题，在两个主要领域，分子识别领域和伴侣行动领域。HdeA非常适合这些研究。它是一个小的10 kDa的内在无序的伴侣，这是高度可重复的NMR结构分析。它与许多具有已知NMR结构的不相关的小客户蛋白形成非常稳定的相互作用，并且HdeA在不存在共分子伴侣或能量源的情况下支持客户重折叠。此外，客户端结合和客户端从HdeA的释放通过简单的pH变化容易且精确地控制。我们将进行详细的NMR残留水平的动态和混乱的HdeA作为pH值的函数的研究，以评估pH值的变化是如何用于控制展开和激活的伴侣。我们将对HdeA和客户蛋白进行残留水平的NMR研究，以同时确定HdeA如何影响其客户蛋白以及客户蛋白如何影响HdeA。我们将使用体内折叠生物传感器直接选择具有改变的灵活性的HdeA突变体，以评估灵活性在伴侣功能中的作用，我们将使用HdeA的客户蛋白免疫蛋白7的选择折叠突变体进行重折叠研究，以确定HdeA如何促进客户重折叠。总之，这些研究将使我们能够表征HdeA分子伴侣活性的分子机制，并测试HdeA分子识别功能所需的灵活性的假设。通过这些研究，我们现在有了一个前所未有的机会来了解分子伴侣的作用机制以及蛋白质功能和分子识别中的障碍作用。

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.