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，可在低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。HdeA的这种应激特异性激活对于大肠杆菌等病原菌在低pH值的胃抗菌环境中生存是必不可少的。HdeA在蛋白质水平上感知压力条件，并以其非常迅速的展开对其做出反应，使HdeA成为一类新的伴侣蛋白的成员，这些伴侣蛋白需要失去结构才能获得活性。最近在其他不依赖于ATP的伴侣蛋白中也有类似的通过部分去折叠的应激特异性激活的报道，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一种新范式。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)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)它支持其客户蛋白在返回非应激条件下重新折叠的机制。这些研究将揭示两个非常普遍的悬而未决的问题的答案：蛋白质如何结合多个无关的客户蛋白，以及伴侣如何与客户蛋白相互作用，以促进它们的折叠。因此，这项工作将 同时解决两个主要领域的基本问题和尚未解决的问题，即分子识别领域和伴侣作用领域。HdeA非常适合于这些研究。它是一个小的10 kDa的本质无序的伴侣，对核磁共振的结构分析有很高的修饰性。它与许多具有已知核磁共振结构的无关小客户蛋白形成非常稳定的相互作用，并且HdeA支持客户在没有辅助伴侣或能源的情况下进行折叠。此外，HdeA的客户结合和客户释放通过简单的pH变化很容易和精确地控制。我们将对HdeA中作为pH函数的动力学和无序进行详细的核磁共振残基水平研究，以评估pH变化如何用于受控展开和激活伴侣蛋白。我们将对HdeA和客户蛋白进行残基水平的核磁共振研究，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灵活性发生变化的HdeA突变体，以评估灵活性在伴侣功能中所起的作用，并将对HdeA的客户蛋白免疫蛋白7的精选折叠突变体进行复性研究，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的伴侣活性背后的分子机制，并测试HdeA的分子识别功能需要灵活性的假设。有了这些研究，我们现在有了一个前所未有的机会，从分子细节上了解伴侣作用的机制以及无序在蛋白质功能和分子识别中的作用。

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.