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

描述(由申请人提供)：本申请的长期目标是从根本上深入了解蛋白质紊乱在伴侣功能中的作用，并更好地了解pH调节的细胞过程。我们专注于细菌伴侣HdeA，它是最近发现的一类压力敏感蛋白的成员，这种蛋白会被导致其展开的条件激活。当致病肠道细菌通过哺乳动物胃的严酷酸性环境时，HdeA经历了pH触发的转变，从不活跃的折叠二聚体转变为伴侣活性的部分未折叠单体，以防止其他未折叠蛋白质在酸诱导的聚集。虽然HdeA活性的一般机制已被了解，但无序单体活性状态的结构细节尚未确定，这阻碍了对其pH胁迫敏感功能的完全了解。通过模拟和实验的多学科相互作用，我们的目标是1)重建作为pH的函数的伴侣活性无序系综，2)表征伴侣与底物的相互作用。我们将进行新的粗粒度模拟来描述HdeA在不同pH环境中的长时间尺度构象动力学。从粗粒度系综出发，我们将建立单体的全原子模型，并针对核磁共振(核磁共振)化学位移和残余偶极耦合进行提炼。基于我们实验精炼的自由伴侣系综，我们将开发一个粗粒度模型来模拟HdeA与核磁共振可访问底物的结合。根据荧光共振、能量转移距离和核磁共振数据，将根据模拟得到的伴侣-底物复合体的结构进行改进。对于实验改进的游离态和结合态HdeA模型，我们将进行全原子恒定pH分子动力学模拟来计算所有酸滴定残基的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触发器”)。我们将通过生化突变研究来测试我们预测的pH触发因素。总的来说，我们的努力将使我们能够充分揭示HdeA在细菌致病中的pH敏感机制，并深入了解伴侣与底物相互作用的方式。

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.