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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 infect 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 becomes partially unfolded. Studies with chemical reducing agents have previously suggested that the intramolecular disulfide bond of HdeA aids in the maintenance of some residual structure at low pH; it is believed that this residual structure is important for clustering 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 mutated the disulfide as conservatively as possible, swapping cysteines with serines. We found that, although residual structure is diminished at low pH without the disulfide, it is not completely lost; conversely, we found that 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, and is better at maintaining the solubility of malate dehydrogenase than chemically-reduced wild type. These studies highlight that we still have much to learn about residual structure at low pH and the role of disulfide bonds, as well as revealing exciting areas of future research.CRediT author statementImex Aguirre-Cardenas: Investigation, Visualization, Formal analysis, Writing – Review and Editing. Dane Geddes-Buehre: Investigation, Formal analysis, Writing – Review and Editing. Karin Crowhurst: Conceptualization, Funding acquisition, Supervision, Project administration, Investigation, Formal analysis, Visualization, Writing – Original Draft, Writing – Review and Editing.

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