Detection of key sites of dimer dissociation and unfolding initiation during activation of acid-stress chaperone HdeA at low pH.
Detection of key sites of dimer dissociation and unfolding initiation during activation of acid-stress chaperone HdeA at low pH.
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在低pH下酸应激伴侣HdeA活化过程中二聚体解离和展开起始的关键位点的检测。
DOI：
10.1016/j.bbapap.2020.140576
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发表时间：
2021-03
期刊：
影响因子：
--
通讯作者：
Crowhurst KA
中科院分区：
文献类型：
--
作者：
Widjaja MA;Gomez JS;Benson JM;Crowhurst KA
HdeA是一种具有独特活性的小分子酸应激伴侣蛋白。在生理pH下，它形成折叠但无活性的二聚体。低于pH 3.0时，HdeA展开并解离成无序单体，利用暴露的疏水补丁结合其他未折叠的蛋白质并防止其不可逆聚集。通过这种方式，HdeA在帮助致病菌在我们的酸性胃中生存并在我们的肠道中定植，促进痢疾的传播方面发挥着关键作用。尽管有许多关于该主题的出版物，但仍存在关于HdeA解折叠和激活的触发机制的问题。以前的研究通常评估HdeA在pH增量下的解折叠，这些pH增量相距太远，无法获得解折叠和二聚体解离过程的细节，并且经常采用阻止对蛋白质特定区域进行彻底评估的技术。我们使用了各种异构体NMR实验，以调查的骨干和侧链的结构和动力学的HdeA在四个pH值之间的变化3.0和2.0。我们发现，在二聚体界面的长环是二聚体解离的起始的早期位点，并且在低pH下，二硫键附近的分子“扣”作为展开的一部分或作为触发器而被打破;该过程还导致C-末端螺旋的分离和关键疏水客户端结合位点的暴露。我们的研究结果突出了HdeA的重要区域，这些区域以前可能被忽视，因为它们离二硫键太近，或者被认为在折叠状态下过于动态，无法影响展开过程。
HdeA is a small acid-stress chaperone protein with a unique activity profile. At physiological pH, it forms a folded, but inactive, dimer. Below pH 3.0, HdeA unfolds and dissociates into disordered monomers, utilizing exposed hydrophobic patches to bind other unfolded proteins and prevent their irreversible aggregation. In this way, HdeA has a key role in helping pathogenic bacteria survive our acidic stomach and colonize our intestines, facilitating the spread of dysentery. Despite numerous publications on the topic, there remain questions about the mechanism by which HdeA unfolding and activation are triggered. Previous studies usually assessed HdeA unfolding over pH increments that are too far apart to gain fine detail of the process of unfolding and dimer dissociation, and often employed techniques that prevented thorough evaluation of specific regions of the protein. We used a variety of heteronuclear NMR experiments to investigate changes to backbone and side chain structure and dynamics of HdeA at four pHs between 3.0 and 2.0. We found that the long loop in the dimer interface is an early site of initiation of dimer dissociation, and that a molecular “clasp” near the disulfide bond is broken open at low pH as part, or as a trigger, of unfolding; this process also results in the separation of C-terminal helices and exposure of key hydrophobic client binding sites. Our results highlight important regions of HdeA that may have previously been overlooked because they lie too close to the disulfide bond or are thought to be too dynamic in the folded state to influence unfolding processes.
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