Structure of the Acinetobacter baumannii Dithiol Oxidase DsbA Bound to Elongation Factor EF-Tu Reveals a Novel Protein Interaction Site

Premkumar, Lakshmanane; Kurth, Fabian; Duprez, Wilko; Grøftehauge, Morten K.; King, G. J.; Halili, Maria A.; Heras, Begoña; Martin, Jennifer L.

doi:10.1074/jbc.m114.571737

Cited by 15 publications

(21 citation statements)

References 84 publications

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“…In Acinetobacter baumannii DsbA (AbDsbA) the groove can accommodate an elongated peptide chain as determined by a crystal structure of AbDsbA in complex with E . coli Elongation Factor EF-Tu [45]. Whilst the physiological relevance of this particular protein-protein interaction remains to be determined, it suggests that the groove may serve as an additional protein interaction site in at least DsbA-I type enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, binding of AbDsbA to E . coli EF-Tu (which engages AbDsbA along an equivalent groove on the non-catalytic face), significantly reduced AbDsbA enzymatic activity in an in vitro model-peptide folding assay [45] suggesting a potential for inhibition by targeting this face of this protein. However, Fragment 1 was found to have no inhibitory activity in the same in vitro model-peptide folding assay even at final concentrations of 10 mM.…”

Section: Discussionmentioning

confidence: 99%

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Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1

et al. 2017

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At a time when the antibiotic drug discovery pipeline has stalled, antibiotic resistance is accelerating with catastrophic implications for our ability to treat bacterial infections. Globally we face the prospect of a future when common infections can once again kill. Anti-virulence approaches that target the capacity of the bacterium to cause disease rather than the growth or survival of the bacterium itself offer a tantalizing prospect of novel antimicrobials. They may also reduce the propensity to induce resistance by removing the strong selection pressure imparted by bactericidal or bacteriostatic agents. In the human pathogen Pseudomonas aeruginosa, disulfide bond protein A (PaDsbA1) plays a central role in the oxidative folding of virulence factors and is therefore an attractive target for the development of new anti-virulence antimicrobials. Using a fragment-based approach we have identified small molecules that bind to PaDsbA1. The fragment hits show selective binding to PaDsbA1 over the DsbA protein from Escherichia coli, suggesting that developing species-specific narrow-spectrum inhibitors of DsbA enzymes may be feasible. Structures of a co-complex of PaDsbA1 with the highest affinity fragment identified in the screen reveal that the fragment binds on the non-catalytic surface of the protein at a domain interface. This biophysical and structural data represent a starting point in the development of higher affinity compounds, which will be assessed for their potential as selective PaDsbA1 inhibitors.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1

et al. 2017

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“…In principle, inhibition of DsbA could lessen the severity of bacterial infections without a selective bactericidal activity that would foster resistance. DsbA proteins have multiple binding interfaces: a hydrophobic patch immediately adjacent to the catalytic CXXC motif, and a non-catalytic groove on the opposite face involved in protein-protein interactions (12,36). Both sites have successfully been targeted with peptide and small molecules.…”

Section: Single Molecule Measurement Of Protein Folding By Magnetic Tmentioning

confidence: 99%

“…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%

“…Anti-DsbA therapeutic development has largely focused on inhibiting its oxidoreductase activity localized to the catalytic hydrophobic groove (10,11,55,56). Recent efforts have also explored potential small molecule and peptide binders of the non-catalytic groove on the opposite face, thought to be involved in protein-protein interactions (12,57). In this context, it is noteworthy that the PWATCDS peptide which binds at the catalytic hydrophobic groove and inhibits the DsbA oxidase also inhibits the mechanical foldase.…”

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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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Structural bioinformatic analysis of DsbA proteins and their pathogenicity associated substrates

Santos-Martin

Wang

Subedi

et al. 2021

Computational and Structural Biotechnology Journal

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Structure of the Acinetobacter baumannii Dithiol Oxidase DsbA Bound to Elongation Factor EF-Tu Reveals a Novel Protein Interaction Site

Cited by 15 publications

References 84 publications

Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1

Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1

DsbA is a redox-switchable mechanical chaperone

Structural bioinformatic analysis of DsbA proteins and their pathogenicity associated substrates

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