Crystal structure of the dithiol oxidase DsbA enzyme from proteus mirabilis bound non-covalently to an active site peptide ligand

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.

Keywords: Crystal Structure; Dithiol Oxidase; Enzyme Catalysis; Enzyme Structure; Oxidative Folding; Peptide Interaction; Protein·Peptide Complex; Structural Biology; Thioredoxin fold; Virulence.

FIGURE 1.

Redox properties of PmDsbA. A, in vitro disulfide catalysis. Plot shows increase in fluorescence as a consequence of peptide disulfide formation catalyzed by EcDsbA or PmDsbA in the presence of EcDsbB. B, in vivo disulfide catalysis. ΔdsbA knock-out or ΔdsbA/B double knock-out cells are non-motile due to their inability to fold FlgI. Expression of PmDsbA or EcDsbA restores motility in ΔdsbA, but not in ΔdsbA/B. C, thermal melting curves of oxidized and reduced PmDsbA shows that reduced PmDsbA (T_m^red 348.4 ± 0.1 K) is more stable than its oxidized counterpart (T_m^ox 338.4 ± 0.2 K). D, measurement of PmDsbA redox potential. PmDsbA was equilibrated in glutathione (GSSG/GSH) redox buffers to measure the equilibrium constant K_eq (187. 5 ± 6 μm), which corresponds to a redox potential of −129 mV. E, absorbance of the catalytic thiolate anion is pH-dependent and this property was used to determine that the pK_a of PmDsbA Cys³⁰ is 4.0. F, disulfide reductase activity measured by following A₆₅₀ nm. PmDsbA has activity similar to that of EcDsbA, and much lower than that of the isomerase EcDsbC. For panels A and C–F, data are presented as mean ± S.D. from three biological replicates. The error bars for the DsbA-only and buffer-only controls in panel A are essentially 0 as there was no increase in fluorescence over the period of the experiment.

FIGURE 2.

Peptide PWATCDS interacts with PmDsbA. A, values of ΔT_m upon addition of increasing PWATCDS peptide for PmDsbA and PmDsbAC30S. Data are shown as mean ± S.D. from 5 replicates. B, ITC data titrating PWATCDS into PmDsbA. The reaction was exothermic suggesting a dominant enthalpic contribution to binding. C, ITC data titrating PWATDCS into PmDsbAC30S. Panels B and C show a representative example from three replicates.

FIGURE 3.

Crystal structure of PmDsbA and its comparison with close homologues. A, structural comparison of four closely related DsbA homologues, PmDsbA (4OCE) in cyan, EcDsbA (1FVK protomer B) in white, KpDsbA (4MCU protomer E) in orange, and SeDsbA in green. Conserved structural regions are annotated; the catalytic cysteines are shown as yellow spheres. B, structure based sequence alignment of EcDsbA (1FVK, chain A), SeDsbA (3L9S, chain A), KpDsbA (4MCU, chain F), and PmDsbA (4OCE, chain A). Sequence identity with EcDsbA is shown on the left, conserved residues are black, differing residues are red. The TRX domain is highlighted in light gray, and the helical domain in dark gray. Red squares mark the three loop regions L1/2/3. Amino acid conservation code is shown beneath the sequences. The active site CXXC motif is highlighted in blue, and the cis-Pro region in green. Secondary structure elements are indicated above the sequences. Residues underlined in EcDsbA bind to the EcDsbB P2 loop peptide (PISA server analysis). C, CXXC active site of PmDsbA. D, CXXC active site of PmDsbAC30S:PWATCDS. For panels C and D, the electron density is from 2F₀ − F_c FFT maps generated in Phenix (53) and contoured at 1.0 σ.

FIGURE 4.

Analysis of the interaction between peptide PWATCDS and PmDsbAC30S. A, superposition of the PWATCDS peptides from all three protomer·peptide complexes in the asymmetric unit. Note the peptide includes an N-terminal acetyl and a C-terminal amide group. Carbon atoms are shown in cyan, oxygens in red, nitrogens in blue, and sulfurs in yellow. B, location of the bound PWATCDS peptide on the surface of PmDsbAC30S (protomer A shown in gray). Peptide residues are labeled and colored as for panel A. The yellow patch indicates the location of Ser³⁰. C, electron density map of PWATCDS (chain F) at the interface with PmDsbAC30S (chain B). The 2F₀ − F_c map was generated in Phenix (53) and is contoured at 1.0 σ. PmDsbAC30S residues forming the binding site are labeled. D, interactions between the peptide (D–F, in cyan) and protein (chains A-C, in gray) are shown: hydrogen bonds are indicated as black dashed lines; a circle highlights the stacking interaction between Trp² and PmDsbAC30S Tyr¹⁷³. E, superposition of the EcDsbB P2 periplasmic loop (PSPFATCD, orange and black letters, PDB code 2ZUP) with PmDsbAC30S-PWATCDS (red letters, backbone in cyan). F, schematic representation of the interactions formed between EcDsbB P2 loop binding to EcDsbA (top) in comparison to PWATCDS binding to PmDsbAC30S (bottom), showing the comparative shift in register. Covalent bonds are shown as black lines, hydrogen bonds are indicated with red dashed lines, and hydrophobic interactions with black dotted lines.