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深入了解蛋白紊乱在伴侣功能中的作用，并更好地理解ph调节的细胞过程。我们关注的是细菌伴侣HdeA，它是最近发现的一类压力感应蛋白的一员，这种蛋白在导致其展开的特定条件下会被激活。当致病性肠道细菌通过哺乳动物胃部的强酸性环境时，HdeA经历了ph触发的从无活性折叠二聚体到伴侣活性部分未折叠单体的转变，以防止其他未折叠蛋白被酸诱导聚集。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胁迫传感功能的全面了解。通过模拟和实验之间的多学科相互作用，我们的目标是1)重建作为pH函数的伴侣-主动无序系综，2)表征伴侣与底物的相互作用。我们将进行新颖的粗粒度模拟来描述不同pH环境下HdeA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，并根据核磁共振（NMR）化学位移和残余偶极耦合对其进行改进。基于我们的实验改进的自由伴侣系综，我们将开发一个粗粒度模型来模拟HdeA与核磁共振可达底物的结合。从模拟中得到的伴侣-底物复合物的结构将根据荧光共振能量转移距离和核磁共振数据进行细化。对于实验改进的自由和束缚HdeA模型，我们将进行全原子恒定pH分子动力学模拟，以计算所有酸滴定残基的pKa。pKa计算将报告特定的pH调制相互作用（“pH触发器”），这些相互作用有助于单体柔韧性和底物相互作用。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在细菌致病性中的ph感应机制，并深入了解伴侣蛋白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.