The complex role of the N-terminus and acidic residues of HdeA as pH-dependent switches in its chaperone function

Pacheco, Sayuri; Widjaja, Marlyn A.; Gomez, Jafaeth S.; Crowhurst, Karin A.; Abrol, Ravinder

doi:10.1016/j.bpc.2020.106406

Cited by 9 publications

(13 citation statements)

References 40 publications

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“…In a trend that is consistent with other experiments we have done [10, 20], the largest changes are on the C-terminal side of position 18 (residues 20 – 25) and the N-terminal side of position 66 (residues 57 – 65). Within the folded structure, portions of these two regions (residues 20 – 25 and 62 – 65) are adjacent to each other; this proximity seems to be maintained in the unfolded state of the wild type, primarily due to the disulfide tether [10]. However, residues 57 – 61 extend along the entire C-terminal half of helix C, far from residues 20 – 25 and far from the site of the removed disulfide in the mutant.…”

Section: Resultssupporting

confidence: 92%

“…As one might expect, the largest chemical shift differences can be found near (but not necessarily at) the mutated cysteines (Figure 2c). In a trend that is consistent with other experiments we have done [1,11], the largest changes are on the C-terminal side of position 18 (residues 20 -25) and the N-terminal side of position 66 (residues 57 -65). Within the folded structure, portions of these two regions (residues 20 -25 and 62 -65) are adjacent to each other; this proximity seems to be maintained in the unfolded state of the wild type, primarily due to the disulfide tether [11].…”

Section: Chemical Shift Differencessupporting

confidence: 92%

“…Additionally, at pH 2.0 the C18S-C66S mutant displays notable β-sheet propensity at the C-terminus, while wild type shows no real secondary structure in that region ( Figure 3 and Table S1 ). The mutant also displays greater β-sheet propensity at the N-terminus at both pHs compared to unfolded wild type, suggesting that the N- and C-termini may have an increased tendency to form semi-stable β-sheet structures with each other, even more than what we observed when modeling the unfolded wild type with MD simulations [10]. In trying to explain the gain of structure in the mutant at pH 2.0 compared to pH 6.0 we wondered whether there is prior experimental evidence that hydrogen bonds are generally stronger at low pHs compared to neutral pH.…”

Section: Resultsmentioning

confidence: 74%

“…Site-directed mutagenesis was executed using the QuikChange Lightning kit from Agilent. HdeA-C18S-C66S was expressed and purified as outlined previously for wild type HdeA [10, 13, 14], with the exception that a Superdex 75 HiLoad 26/600 column was required rather than the usual HR 10/30 column. All samples were uniformly 13 C/ 15 N labeled.…”

Section: Methodsmentioning

confidence: 99%

“…It has been conjectured that it may help to bring two hydrophobic sites into close contact at low pH, thereby facilitating chaperone activity by enabling the formation of an extended client binding site [9]. Although its importance is mentioned in several publications on HdeA [8,[10][11][12], relatively little investigation has been done on the C18-C66 disulfide bond. Hong et al [8] originally reported that the chaperone activity of HdeA was unaffected by a reduction of the disulfide bond at low pH, but because they utilized dithiothreitol (DTT), which is functional only in the pH range 6.5 -9.0, these observations were not valid.…”

Section: Introductionmentioning

confidence: 99%

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Removal of disulfide from acid stress chaperone HdeA does not wholly eliminate structure or function at low pH

Aguirre-Cardenas

Geddes-Buehre

Crowhurst

2021

Preprint

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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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Section: Resultssupporting

confidence: 92%