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

Aguirre-Cardenas, M. Imex; Geddes-Buehre, Dane H.; Crowhurst, Karin A.

doi:10.1016/j.bbrep.2021.101064

Cited by 5 publications

(7 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Various reports are already available explaining the role of disulfide bonds on the chaperone action of HdeA. 13,15,17,27,29,30 The C18−C66 disulfide bond in HdeA binds its two hydrophobic regions together, essential for its chaperone activity. 18 On the contrary, no investigation has been done on C10−C58 disulfide bond of HdeB.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…The effect of disulfide bonds on the stability of many globular proteins, such as ribonuclease and lysozyme, , has been extensively studied. Earlier studies suggested the effect of disulfide bonds for the maintenance of the partially folded structure and, hence, the chaperone activity of HdeA. ,− , However, the role of the disulfide bond in the conformational stability and chaperone function of HdeB is not known. Herein, we have compared the role of the disulfide bond in the stability of acid-stress chaperones.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

The Chaperone-Active State of HdeB at pH 4 Arises from Its Conformational Rearrangement and Enhanced Stability Instead of Surface Hydrophobicity

Thapliyal,

Mishra

2024

Biochemistry

View full text Add to dashboard Cite

HdeA and HdeB are dimeric ATP-independent acidstress chaperones, which protect the periplasmic proteins of enteric bacteria at pH 2.0 and 4.0, respectively, during their passage through the acidic environment of the mammalian stomach. Despite being structurally similar, they exhibit distinct functional pH optima and conformational prerequisite for their chaperone action. HdeA undergoes a dimer-to-monomer transition at pH 2.0, whereas HdeB remains dimeric at pH 4.0. The monomerization of HdeA exposes its hydrophobic motifs, which facilitates its interaction with the partially folded substrates. How HdeB functions despite maintaining its dimeric conformation has been poorly elucidated in the literature. Herein, we characterized the conformational states and stability of HdeB at its physiologically relevant pH and compared the data with those of HdeA. At pH 4.0, HdeB exhibited distinct spectroscopic signatures and higher stability against heat and guanidine-HCl-induced denaturation than at pH 7.5. We affirm that the pH 4.0 conformer of HdeB was distinct from that at pH 7.5 and that these two conformational states were hierarchically unrelated. Salt-bridge mutations that perturbed HdeB's intersubunit interactions resulted in the loss of its stability and function at pH 4.0. In contrast, mutations affecting intrasubunit interactions enhanced its function, albeit with a reduction in stability. These findings suggest that, unlike HdeA, HdeB acts as a noncanonical chaperone, where pH-dependent stability and conformational rearrangement at pH 4.0 play a core role in its chaperone function rather than its surface hydrophobicity. Such rearrangement establishes a stability-function trade-off that allows HdeB to function while maintaining its stable dimeric state.

show abstract

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

The Chaperone-Active State of HdeB at pH 4 Arises from Its Conformational Rearrangement and Enhanced Stability Instead of Surface Hydrophobicity

Thapliyal,

Mishra

2024

Biochemistry

View full text Add to dashboard Cite

show abstract

“…Subunits of HdeA and HdeB both possess a single disulfide bond in its native state; in the case of HdeB, this disulfide bond is formed between Cys10 and Cys58 of the mature (signal-processed) sequence [ 4 ]. In the case of HdeA, reduction of the disulfide bond causes a large decrease [ 14 ] (but apparently not a complete loss [ 15 ]) of molecular chaperone activity, suggesting that the acid denatured structure is perturbed significantly by the loss of the disulfide bond. Although analogous data regarding the role of the disulfide bond on the structure and function of HdeB are not available, we wished to see if removing this bond would result in a more extensively unfolded, dynamic polypeptide whose fibril forming tendencies would be altered.…”

Section: Resultsmentioning

confidence: 99%

Formation of Fibrils by the Periplasmic Molecular Chaperone HdeB from Escherichia coli

Nakata

Kitazaki

Kanaoka

et al. 2022

IJMS

View full text Add to dashboard Cite

The molecular chaperones HdeA and HdeB of the Escherichia coli (E. coli) periplasm protect client proteins from acid denaturation through a unique mechanism that utilizes their acid denatured states to bind clients. We previously demonstrated that the active, acid-denatured form of HdeA is also prone to forming inactive, amyloid fibril-like aggregates in a pH-dependent, reversible manner. In this study, we report that HdeB also displays a similar tendency to form fibrils at low pH. HdeB fibrils were observed at pH < 3 in the presence of NaCl. Similar to HdeA, HdeB fibrils could be resolubilized by a simple shift to neutral pH. In the case of HdeB, however, we found that after extended incubation at low pH, HdeB fibrils were converted into a form that could not resolubilize at pH 7. Fresh fibrils seeded from these “transformed” fibrils were also incapable of resolubilizing at pH 7, suggesting that the transition from reversible to irreversible fibrils involved a specific conformational change that was transmissible through fibril seeds. Analyses of fibril secondary structure indicated that HdeB fibrils retained significant alpha helical content regardless of the conditions under which fibrils were formed. Fibrils that were formed from HdeB that had been treated to remove its intrinsic disulfide bond also were incapable of resolubilizing at pH 7, suggesting that certain residual structures that are retained in acid-denatured HdeB are important for this protein to recover its soluble state from the fibril form.

show abstract

“…25,29 A conserved disulfide links the only two Cys residues (18 and 66) in the native sequence; its reduction disorders the structure and inhibits chaperone activity in vitro , 28,30 though not entirely. 31 In-vivo consequences of a lost or shifted disulfide have not been investigated; we report that dose-dependent toxicity typically results.…”

Section: Introductionmentioning

confidence: 84%

“…That HdeA folds up when inactive suggests the unfolded state may have a fitness cost, although an in-vivo crosslinking study questioned whether the inactive state is stably folded (Fu et al, 2019). A conserved disulfide links the only two Cys residues (18 and 66) in the native sequence; its reduction disorders the structure and inhibits chaperone activity in vitro (Tapley et al, 2009; Zhai et al, 2016), though not entirely (Aguirre-Cardenas et al, 2021). In-vivo consequences of a lost or shifted disulfide have not been investigated; we now report that dose-dependent cytotoxicity typically results.…”

Section: Introductionmentioning

confidence: 99%

Systematic conformation-to-phenotype mapping via limited deep-sequencing of proteins

Serebryany¹,

Zhao²,

Park³

et al. 2022

Preprint

View full text Add to dashboard Cite

SummaryNon-native conformations drive protein misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well-suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a limited deep-sequencing method for proteins that identifies the exact sequence positions of both cysteines simultaneously within each polypeptide molecule. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments.

show abstract

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

Cited by 5 publications

References 20 publications

The Chaperone-Active State of HdeB at pH 4 Arises from Its Conformational Rearrangement and Enhanced Stability Instead of Surface Hydrophobicity

The Chaperone-Active State of HdeB at pH 4 Arises from Its Conformational Rearrangement and Enhanced Stability Instead of Surface Hydrophobicity

Formation of Fibrils by the Periplasmic Molecular Chaperone HdeB from Escherichia coli

Systematic conformation-to-phenotype mapping via limited deep-sequencing of proteins

Contact Info

Product

Resources

About