Aguirre-Cardenas M Imex, Geddes-Buehre Dane H, Crowhurst Karin A
Department of Chemistry and Biochemistry, California State University Northridge, 18111 Nordhoff St., Northridge, CA, 91330-8262, USA.
Present address: Department of Chemistry, University of California Riverside, 900 University Ave, Riverside, CA, 92521, USA.
Biochem Biophys Rep. 2021 Jul 1;27:101064. doi: 10.1016/j.bbrep.2021.101064. eCollection 2021 Sep.
HdeA is an acid-stress chaperone that operates in the periplasm of various strains of pathogenic gram-negative bacteria. Its primary function is to prevent irreversible aggregation of other periplasmic proteins when the bacteria enter the acidic environment of the stomach after contaminated food is ingested; its role is therefore to help the bacteria survive long enough to enter and colonize the intestines. The mechanism of operation of HdeA is unusual in that this helical homodimer is inactive when folded at neutral pH but becomes activated at low pH after the dimer dissociates and partially unfolds. Studies with chemical reducing agents previously suggested that the intramolecular disulfide bond is important for maintaining residual structure in HdeA at low pH and may be responsible for positioning exposed hydrophobic residues together for the purpose of binding unfolded client proteins. In order to explore its role in HdeA structure and chaperone function we performed a conservative cysteine to serine mutation of the disulfide. We found that, although residual structure is greatly diminished at pH 2 without the disulfide, it is not completely lost; conversely, the mutant is almost completely random coil at pH 6. Aggregation assays showed that mutated HdeA, although less successful as a chaperone than wild type, still maintains a surprising level of function. These studies highlight that we still have much to learn about the factors that stabilize residual structure at low pH and the role of disulfide bonds.
HdeA是一种酸性应激伴侣蛋白，在多种革兰氏阴性病原菌的周质中发挥作用。其主要功能是在细菌摄入受污染食物后进入胃部酸性环境时，防止其他周质蛋白发生不可逆聚集；因此，它的作用是帮助细菌存活足够长的时间以进入肠道并在其中定殖。HdeA的作用机制不同寻常，这种螺旋同型二聚体在中性pH值下折叠时无活性，但在低pH值下二聚体解离并部分展开后会被激活。先前使用化学还原剂进行的研究表明，分子内二硫键对于在低pH值下维持HdeA中的残余结构很重要，并且可能负责将暴露的疏水残基定位在一起，以便结合未折叠的客户蛋白。为了探究其在HdeA结构和伴侣功能中的作用，我们对二硫键进行了保守的半胱氨酸到丝氨酸突变。我们发现，虽然在没有二硫键的情况下，pH 2时残余结构大大减少，但并未完全丧失；相反，突变体在pH 6时几乎完全是无规卷曲。聚集分析表明，突变的HdeA虽然作为伴侣蛋白不如野生型成功，但仍保持着惊人的功能水平。这些研究突出表明，我们对于在低pH值下稳定残余结构的因素以及二硫键的作用仍有很多需要了解的地方。
