The Role of Conditional Disorder in the Acid Stress-Sensing Chaperone HdeA

条件性障碍在酸应激感应伴侣 HdeA 中的作用

• 批准号: 8710539
• 负责人: Logan S Ahlstrom
• 金额: $ 5.15万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8710539
• 关键词: Accounting; Acids; Address; Binding; Biochemical; Biochemistry; Biological Assay; Biomolecular Nuclear Magnetic Resonance; Cell physiology; Cereals; Charge; Chemicals; Collaborations; Complex; Data; Disease; Dissociation; Enterobacteriaceae; Environment; Equilibrium; Event; Exhibits; Experimental Designs; Fluorescence Resonance Energy Transfer; Goals; Health; Immunity; Invaded; Methods; Michigan; Modeling; Molecular; Molecular Chaperones; Nature; Nuclear Magnetic Resonance; Pathogenicity; Periplasmic Proteins; Play; Protein Dynamics; Proteins; Relative (related person); Reporting; Research; Residual state; Resistance; Role; Sampling; Simulate; Specialist; Stomach; Stress; Structure; Substrate Interaction; Testing; Time; Universities; Work; acid stress; base; conformer; design; dimer; flexibility; fly; gain of function; insight; member; microorganism; models and simulation; molecular dynamics; monomer; multi-scale modeling; mutant; novel; prevent; protein function; protein structure; protonation; research study; response; sensor; simulation

DESCRIPTION (provided by applicant): The long-term goals of this application are to yield fundamental insight into the role of protein disorder in chaperone function and to better understand pH-modulated cellular processes. We focus on the bacterial chaperone HdeA, which is a member of a recently discovered class of stress-sensing proteins that become activated by the very conditions that cause their unfolding. As pathogenic enteric bacteria pass through the harshly acidic environment of the mammalian stomach, HdeA undergoes a pH-triggered transition from an inactive folded dimer to a chaperone-active partially unfolded monomer to prevent other unfolded proteins from acid-induced aggregation. Although a general mechanism for HdeA activity is understood, the structural details of the disordered monomeric active state have yet to be determined, preventing a complete understanding of its pH stress-sensing function. Through a multi-disciplinary interplay between simulation and experiment, we aim to 1) reconstruct the chaperone-active disordered ensemble as a function of pH and 2) characterize chaperone interaction with substrate. We will perform novel coarse-grained simulations to describe the long time-scale conformational dynamics of HdeA in different pH environments. From the coarse-grained ensemble, we will build all-atom models of the monomer and refine them against nuclear magnetic resonance (NMR) chemical shifts and residual dipolar couplings. Based on our experimentally refined ensemble of free chaperone, we will develop a coarse-grained model to simulate HdeA bound to an NMR-accessible substrate. The structure of the chaperone-substrate complex from simulation will be refined against fluorescence resonance energy transfer distances and NMR data. For the experimentally refined models of free and bound HdeA, we will perform all-atom constant pH molecular dynamics simulation to calculate the pKa's of all acid titratable residues. The pKa calculations will report on specific pH-modulated interactions ("pH triggers") that contribute to monomer flexibility and substrate interaction. We will test our predicted pH triggers through biochemical mutational studies. Collectively, our efforts will allow us to fully uncover the pH-sensing mechanism of HdeA in bacterial pathogenicity and to gain insight into the manner in which chaperones interact with substrate.

描述（由申请人提供）：本申请的长期目标是对蛋白质紊乱在分子伴侣功能中的作用产生基本的认识，并更好地理解pH调节的细胞过程。我们专注于细菌分子伴侣HdeA，这是最近发现的一类压力传感蛋白的成员，这些蛋白被导致其展开的条件激活。当致病性肠道细菌通过哺乳动物胃的苛刻酸性环境时，HdeA经历pH触发的从无活性折叠二聚体到分子伴侣活性部分未折叠单体的转变，以防止其他未折叠蛋白质由酸诱导的聚集。虽然HdeA活性的一般机制是理解的，无序的单体活性状态的结构细节尚未确定，防止其pH应力传感功能的完整理解。通过模拟和实验之间的多学科相互作用，我们的目标是1）重建分子伴侣活性无序系综作为pH值的函数和2）表征分子伴侣与底物的相互作用。我们将进行新的粗粒度的模拟来描述在不同的pH值环境中的长时间尺度的构象动力学的HdeA。从粗粒系综，我们将建立单体的全原子模型，并对核磁共振（NMR）化学位移和残余偶极耦合进行改进。基于我们的实验精炼合奏的自由分子伴侣，我们将开发一个粗粒度的模型来模拟HdeA绑定到NMR可访问的基板。来自模拟的分子伴侣-底物复合物的结构将针对荧光共振能量转移距离和NMR数据进行细化。对于自由和结合HdeA的实验改进模型，我们将进行全原子恒定pH分子动力学模拟，以计算所有酸可滴定残基的pKa。pKa计算将报告有助于单体灵活性和底物相互作用的特定pH调节相互作用（“pH触发物”）。我们将通过生化突变研究来测试我们预测的pH触发器。总的来说，我们的努力将使我们能够充分揭示HdeA在细菌致病性中的pH敏感机制，并深入了解分子伴侣与底物相互作用的方式。