Mechanism of an Acid Activated Chaperone

酸激活伴侣的机制

• 批准号: 8502896
• 负责人: Hashim M Al-Hashimi
• 金额: $ 48.42万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8502896
• 关键词: Acids; Address; Affect; Bacteria; Bacterial Proteins; Binding; Binding Proteins; Biochemical; Biosensor; Client; Complex; Digestion; Dimerization; Disease; Dissociation; Electrostatics; Energy-Generating Resources; Environment; Escherichia coli; Food; Immunity; Ingestion; Kinetics; Link; Mediating; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Monitor; Oral; Periplasmic Proteins; Play; Property; Protein Binding; Proteins; Reporting; Resolution; Role; Sequence Alignment; Specificity; Stomach; Stress; Structure; Techniques; Testing; Time; Variant; Work; antimicrobial; cofactor; disulfide bond; flexibility; in vivo; innovation; insight; killings; member; molecular dynamics; molecular recognition; monomer; mutant; pathogenic bacteria; prevent; protein aggregation; protein complex; protein folding; protein function; public health relevance; research study; tool

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.

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 条件下快速激活，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胃的低 pH 抗菌环境中生存至关重要。 HdeA 能够感知蛋白质水平的应激条件，并通过自身非常快速的展开对其做出反应，这使得 HdeA 成为新型伴侣蛋白的一员，这种伴侣蛋白需要失去结构才能获得活性。最近也报道了其他不依赖于 ATP 的分子伴侣通过部分解折叠实现的类似应激特异性激活，这表明这种机制可能代表了分子伴侣和本质无序蛋白质领域的新范例。我们现在将使用结构、生化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) 它的机制支持其客户蛋白在回到非应激条件下的重折叠。这些研究将揭示两个非常普遍的未解答问题的答案：蛋白质如何结合多个不相关的客户蛋白，以及伴侣如何与客户蛋白相互作用以促进其重折叠。因此这部作品将 同时解决分子识别领域和分子伴侣作用领域两个主要领域中尚未解决的基本问题。 HdeA 非常适合这些研究。它是一种小的 10 kDa 本质上无序的分子伴侣，非常适合通过 NMR 进行结构分析。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 在没有共伴侣或能源的情况下支持客户重折叠。此外，通过简单的 pH 变化可以轻松、精确地控制 HdeA 的客户结合和客户释放。我们将对 HdeA 中的动力学和无序作为 pH 函数进行详细的 NMR 残留水平研究，以评估 pH 变化如何用于伴侣蛋白的受控展开和激活。我们将对 HdeA 和客户蛋白进行残留水平 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，并且我们将使用HdeA的客户蛋白免疫蛋白7的精选折叠突变体进行重折叠研究，以确定HdeA如何促进客户蛋白重新折叠。总之，这些研究将使我们能够表征 HdeA 伴侣活性的分子机制，并检验 HdeA 分子识别功能需要灵活性的假设。通过这些研究，我们现在有一个前所未有的机会在分子细节上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紊乱的作用。