Abstract
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.