Wilko Duprez

Rv2969c, essential for optimal growth in Mycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases.

The gene product of M. tuberculosis Rv2969c is shown to be a disulfide oxidase enzyme that has a canonical DsbA-like fold with novel structural and functional characteristics.

Peptide Inhibitors of the Escherichia coli DsbA Oxidative Machinery Essential for Bacterial Virulence

One approach to address antibiotic resistance is to develop drugs that interfere with bacterial virulence. A master regulator of virulence in Gram-negative bacteria is the oxidative folding machinery comprising DsbA and DsbB. A crystal structure at 2.5 Å resolution is reported here for Escherichia coli DsbA complexed with PFATCDS, a heptapeptide derived from the partner protein Escherichia coli DsbB. Details of the peptide binding mode and binding site provide valuable clues for inhibitor design. Structure-activity relationships for 30 analogues were used to produce short peptides with a cysteine that bind tightly to EcDsbA (Kd = 2.0 ± 0.3 μM) and inhibit its activity (IC50 = 5.1 ± 1.1 μM). The most potent inhibitor does not bind to or inhibit human thioredoxin that shares a similar active site. This finding suggests that small molecule inhibitors can be designed to exploit a key interaction of EcDsbA, as the basis for antivirulence agents with a novel mechanism of action.

Comparative Sequence, Structure and Redox Analyses of Klebsiella pneumoniae DsbA Show That Anti-Virulence Target DsbA Enzymes Fall into Distinct Classes

Bacterial DsbA enzymes catalyze oxidative folding of virulence factors, and have been identified as targets for antivirulence drugs. However, DsbA enzymes characterized to date exhibit a wide spectrum of redox properties and divergent structural features compared to the prototypical DsbA enzyme of Escherichia coli DsbA (EcDsbA). Nonetheless, sequence analysis shows that DsbAs are more highly conserved than their known substrate virulence factors, highlighting the potential to inhibit virulence across a range of organisms by targeting DsbA. For example, Salmonella enterica typhimurium (SeDsbA, 86 % sequence identity to EcDsbA) shares almost identical structural, surface and redox properties. Using comparative sequence and structure analysis we predicted that five other bacterial DsbAs would share these properties. To confirm this, we characterized Klebsiella pneumoniae DsbA (KpDsbA, 81 % identity to EcDsbA). As expected, the redox properties, structure and surface features (from crystal and NMR data) of KpDsbA were almost identical to those of EcDsbA and SeDsbA. Moreover, KpDsbA and EcDsbA bind peptides derived from their respective DsbBs with almost equal affinity, supporting the notion that compounds designed to inhibit EcDsbA will also inhibit KpDsbA. Taken together, our data show that DsbAs fall into different classes; that DsbAs within a class may be predicted by sequence analysis of binding loops; that DsbAs within a class are able to complement one another in vivo and that compounds designed to inhibit EcDsbA are likely to inhibit DsbAs within the same class.

Membrane-curvature protein exhibits interdomain flexibility and binds a small GTPase

Background: APPL2 is an endosomal Rab effector forming part of a signaling pathway linking cell surface and nucleus. Results: Crystal and solution structures of APPL2 were solved, and Rab partners were identified. Conclusion: APPL2 interacts tightly with Rab31, and APPL2 structures reveal unexpected domain motion that could have functional implications. Significance: APPL2 dynamics and interactions may be crucial for its cell signaling role. The APPL1 and APPL2 proteins (APPL (adaptor protein, phosphotyrosine interaction, pleckstrin homology (PH) domain, and leucine zipper-containing protein)) are localized to their own endosomal subcompartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus. They play important roles in signal transduction through their ability to act as Rab effectors. (Rabs are a family of Ras GTPases involved in membrane trafficking.) Both APPL1 and APPL2 comprise an N-terminal membrane-curving BAR (Bin-amphiphysin-Rvs) domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain. The structure and interactions of APPL1 are well characterized, but little is known about APPL2. Here, we report the crystal structure and low resolution solution structure of the BARPH domains of APPL2. We identify a previously undetected hinge site for rotation between the two domains and speculate that this motion may regulate APPL2 functions. We also identified Rab binding partners of APPL2 and show that these differ from those of APPL1, suggesting that APPL-Rab interaction partners have co-evolved over time. Isothermal titration calorimetry data reveal the interaction between APPL2 and Rab31 has a Kd of 140 nm. Together with other biophysical data, we conclude the stoichiometry of the complex is 2:2.

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

Background: DsbA enzymes assemble bacterial virulence factors and are targets for an entirely new drug class. Results: Proteus mirabilis DsbA was characterized and its structure determined with a peptide bound non-covalently at the active site. Conclusion: The structure provides an important basis for future inhibitor design. Significance: New drugs to treat superbugs are urgently needed. DsbA inhibitors could have antivirulence activity against bacterial pathogens. 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.

Virtual Screening of Peptide and Peptidomimetic Fragments Targeted to Inhibit Bacterial Dithiol Oxidase DsbA.

Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB - or peptides - complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.

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

Background: DsbA is a master virulence determinant of bacterial pathogens and a target for antivirulence drugs. Results: AbDsbA is a class I dithiol oxidase that binds EF-Tu-derived and DsbB-derived peptides on different enzyme surfaces. Conclusion: Discovery of high affinity peptide interaction sites provides a platform for inhibitor design. Significance: AbDsbA inhibitors could have anti-biofilm activity against multidrug resistant Acinetobacter baumannii. The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA.

Structure of theAcinetobacter baumanniiDithiol Oxidase DsbA Bound to Elongation Factor EF-Tu Reveals a Novel Protein Interaction Site

Background: DsbA is a master virulence determinant of bacterial pathogens and a target for antivirulence drugs. Results: AbDsbA is a class I dithiol oxidase that binds EF-Tu-derived and DsbB-derived peptides on different enzyme surfaces. Conclusion: Discovery of high affinity peptide interaction sites provides a platform for inhibitor design. Significance: AbDsbA inhibitors could have anti-biofilm activity against multidrug resistant Acinetobacter baumannii. The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA.

Structure of the Acinetobacter baumannii dithiol oxidase DsbA bound to EF-Tu reveals a novel protein interaction site

Background: DsbA is a master virulence determinant of bacterial pathogens and a target for antivirulence drugs. Results: AbDsbA is a class I dithiol oxidase that binds EF-Tu-derived and DsbB-derived peptides on different enzyme surfaces. Conclusion: Discovery of high affinity peptide interaction sites provides a platform for inhibitor design. Significance: AbDsbA inhibitors could have anti-biofilm activity against multidrug resistant Acinetobacter baumannii. The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA.