Abstract
The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery.
Highlights
DsbA enzymes assemble bacterial virulence factors and are targets for an entirely new drug class
The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens
PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA
Summary
DsbA enzymes assemble bacterial virulence factors and are targets for an entirely new drug class. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. DsbA enzymes are thioredoxin-fold proteins [26] localized to the periplasm of Gram-negative bacteria where they catalyze oxidative folding [27] This reaction involves the transfer of a disulfide bond from the active site 30CXXC33 motif of DsbA to cysteine thiols in newly translocated proteins [28]. We report three high-resolution crystal structures including the PmDsbAC30S1⁄7PWATCDS complex, which represents to our knowledge the first reported example of a peptide bound non-covalently to a DsbA structure
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