Crystal Structure of the Dithiol Oxidase DsbA Enzyme from Proteus Mirabilis Bound Non-covalently to an Active Site Peptide Ligand

Kurth, Fabian; Duprez, Wilko; Premkumar, Lakshmanane; Schembri, Mark A.; Fairlie, David P.; Martin, Jennifer L.

doi:10.1074/jbc.m114.552380

Cited by 22 publications

(43 citation statements)

References 76 publications

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“…Although PDI obtained near-unity amplitude as well, the fractional occurrence of reduced domains due to DsbA activity levels off at 0.44. We cite this observation as evidence of non-covalent interactions between unfolded substrate and enzyme, which have been described previously by others but remain poorly understood (17). The non-covalent interactions stabilize a previously unconsidered state in which the disulfide has returned to the enzyme but unbinding and diffusion have not yet occurred.…”

Section: Figurementioning

confidence: 82%

“…We note an especially striking disparity in the kinetics of non-oxidative release, a process that occurs with relative ease for PDI but is largely suppressed by DsbA. In a novel and expanded mechanistic model, we suggest that this arises from surprisingly strong enzyme-substrate non-covalent interactions, which have recently been documented but remain largely unexplained (17).…”

mentioning

confidence: 97%

“…In various pathogenic bacteria, DsbA deletion results in a removal or significant reduction in virulence or pathogenicity (1)(2)(3)(12)(13)(14)(15)(16). Because of this, DsbA has potential as a target for novel antibiotics to combat the increasingly urgent issue of antibiotic-resistant pathogens (17).…”

mentioning

confidence: 99%

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Monitoring Oxidative Folding of a Single Protein Catalyzed by the Disulfide Oxidoreductase DsbA

Kahn

Fernández

Pérez-Jiménez

2015

Journal of Biological Chemistry

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Background:The enzyme DsbA is essential for production of disulfide-bonded proteins in E. coli. Results: The folding, misfolding, and release kinetics of a substrate interacting with DsbA are determined from single molecule observations. Conclusion: DsbA is much more effective than its eukaryotic counterpart. Significance: Previous mechanistic models are generalized while providing insight into the kinetic parameters that influence oxidative folding outcomes.

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Section: Figurementioning

confidence: 82%

mentioning

confidence: 97%

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Monitoring Oxidative Folding of a Single Protein Catalyzed by the Disulfide Oxidoreductase DsbA

Kahn

Fernández

Pérez-Jiménez

2015

Journal of Biological Chemistry

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“…Interestingly most of these translocated extracellular proteins in bacteria contain disulphide bonds which are introduced by oxidoreductase enzymes of the Dsb-family that share a conserved thioredoxin-type fold (9). The prototypical family member DsbA is necessary for the maturation of a range of virulence factors including flagellar motors and pilus adhesins, Type III secretion systems, and heat-labile and heat-stable enterotoxins and has emerged as a target for novel antibiotic development (10)(11)(12)(13). Notably, DsbA is an ongoing target for drug development due to its direct relevance in urinary tract infections, bacteremia, and biological weapons development (13)(14)(15)(16).…”

Section: Introductionmentioning

confidence: 99%

DsbA is a redox-switchable mechanical chaperone

Eckels¹,

Chaudhuri²,

Chakraborty³

et al. 2018

Preprint

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Force is a crucial protein denaturant in cells, occurring during processes including translation, degradation, and translocation. In bacteria, many toxins and virulence factors pass through a translocon pore as unfolded polypeptides en route to periplasmic and extracellular compartments.DsbA is a ubiquitous bacterial oxidoreductase that associates with substrates during and after translocation, yet its involvement in protein folding and translocation remains an open question.Here, using magnetic tweezers-based single molecule force spectroscopy, we characterize a mechanical chaperone activity of DsbA on a cysteine-free domain from the protein L superantigen. Interaction of DsbA with unfolded protein L substrates prominently increases the fraction of time that protein L spends in its native folded state. This chaperone activity is tuned by the oxidation state of DsbA; oxidized DsbA is a strong promoter of folding, but the effect is weakened by reduction of the catalytic CXXC motif. We further localize the chaperone binding site of DsbA using a seven residue peptide which effectively blocks the chaperone activity. We calculated that DsbA assisted folding of proteins in the periplasm generates enough mechanical work to decrease the ATP consumption needed for periplasmic translocation by up to 33%. In turn, pharmacologic inhibition of this chaperone activity may open up a new class of antivirulence agents.

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“…The inhibitory binding of EFTu to the non-catalytic surface of AbDsbA [82] presents the possibility of developing allosteric inhibitors that don't bind to the catalytic surface. This is a promising strategy for development of drugs for proteins such as CtDsbA that have no apparent pocket at the catalytic surface, but two pockets on the posterior surface.…”

Section: Future Perspectives For Developing Antimicrobial Drugs Targementioning

confidence: 99%

The disulfide oxidative pathway in Chlamydia trachomatis

Christensen¹

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The disulfide oxidative pathway in bacteria is responsible for disulfide bond formation in secreted proteins including many virulence factors. The archetypal Escherichia coli DsbA and DsbB enzymes form a redox relay that catalyses disulfide bond formation and constitutes the oxidative folding pathway. DsbA orthologs from a broad range of bacteria have been characterised and while the enzymes have similar structural and biochemical characteristics they vary in redox chemistry and surface properties. An overview of the DsbA and DsbB enzymes from different bacteria is provided in Chapter 1. Chapter 1 also provides an overview of antibiotics with well-known and novel targets, including bacterial oxidative folding.Chlamydia trachomatis is an obligate intracellular pathogen responsible for blinding trachoma and the sexually transmitted infection chlamydia that can lead to infertility and ectopic pregnancy. C. trachomatis has a biphasic developmental cycle that is dependent on coordinated disulfide bond formation and reduction for differentiation between the two distinct cell types: reticular body (RB) and elementary body (EB) (reviewed in chapter 1).Chapter 2 presents a biochemical and structural characterisation of a truncated soluble form of DsbA from C. trachomatis (CtDsbA), which was published in PLOS ONE. CtDsbA is the first structurally characterised DsbA having two small, uncharged amino acids separating the two active site cysteines (Cys-Ser-Ala-Cys). Characterisation of CtDsbA shows that is has oxidase activity and is structurally similar to other DsbAs. However, CtDsbA is distinct in that it is the weakest oxidising DsbA so far described. This is consistent with the analysis of the 2.7 Å resolution crystal structure showing a lack of factors stabilising the nucleophilic thiolate anion of Cys38. CtDsbA is also distinguished from most other DsbAs by having a disulfide bond, linking helix 2 and helix 5, that has only been reported in three other structurally characterised DsbAs, including Wolbachia pipientis DsbA1 (WpDsbA1).Chapter 3 describes my studies of the interaction between CtDsbA and CtDsbB. In the oxidative pathway of E. coli, DsbA (EcDsbA) is kept in its active, oxidised state by the integral membrane protein DsbB (EcDsbB) in a mechanism dependent on a quinone cofactor. A BLAST search identified a protein (CtDsbB) in the genome of C. trachomatis with 21% sequence identity to EcDsbB and a predicted secondary structure topology equivalent to EcDsbB. Chapter 3 presents the characterisation of the interaction between CtDsbA and CtDsbB showing that crude membranes containing heterogeneously expressed CtDsbB are able to oxidize CtDsbA. However, purified, detergent solubilised CtDsbB is not able to oxidise CtDsbA in the presence of ubiquinone-1 suggesting that purified CtDsbB is inactive in detergent micelles or that ubiquinone-1 is not a suitable cofactor for CtDsbB. In contrast to what was found for Wolbachia DsbA1, the non-catalytic disulfide of CtDsbA does not regulate interaction with CtDsbB. Interesting...

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Crystal Structure of the Dithiol Oxidase DsbA Enzyme from Proteus Mirabilis Bound Non-covalently to an Active Site Peptide Ligand

Cited by 22 publications

References 76 publications

Monitoring Oxidative Folding of a Single Protein Catalyzed by the Disulfide Oxidoreductase DsbA

Monitoring Oxidative Folding of a Single Protein Catalyzed by the Disulfide Oxidoreductase DsbA

DsbA is a redox-switchable mechanical chaperone

The disulfide oxidative pathway in Chlamydia trachomatis

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