A Buried Water Network Modulates the Activity of the Escherichia coli Disulphide Catalyst DsbA.

The formation of disulphide bonds is an essential step in the folding of many proteins that enter the secretory pathway; therefore, it is not surprising that eukaryotic and prokaryotic organisms have dedicated enzymatic systems to catalyse this process. In bacteria, one such enzyme is disulphide bond-forming protein A (DsbA), a thioredoxin-like thiol oxidase that catalyses the oxidative folding of proteins required for virulence and fitness. A large body of work on DsbA proteins, particularly Escherichia coli DsbA (EcDsbA), has demonstrated the key role that the Cys30-XX-Cys33 catalytic motif and its unique redox properties play in the thiol oxidase activity of this enzyme. Using mutational and functional analyses, here we identify that a set of charged residues, which form an acidic groove on the non-catalytic face of the enzyme, further modulate the activity of EcDsbA. Our high-resolution structures indicate that these residues form a water-mediated proton wire that can transfer protons from the bulk solvent to the active site. Our results support the view that proton shuffling may facilitate the stabilisation of the buried Cys33 thiolate formed during the redox reaction and promote the correct direction of the EcDsbA-substrate thiol-disulphide exchange. Comparison with other proteins of the same class and proteins of the thioredoxin-superfamily in general suggest that a proton relay system appears to be a conserved catalytic feature among this widespread superfamily of proteins. Furthermore, this study also indicates that the acidic groove of DsbA could be a promising allosteric site to develop novel DsbA inhibitors as antibacterial therapeutics.

Methyl probes in proteins for determining ligand binding mode in weak protein-ligand complexes.

Structures of protein-ligand complexes provide critical information for drug design. Most protein-ligand complex structures are determined using X-ray crystallography, but where crystallography is not able to generate a structure for a complex, NMR is often the best alternative. However, the available tools to enable rapid and robust structure determination of protein-ligand complexes by NMR are currently limited. This leads to situations where projects are either discontinued or pursued without structural data, rendering the task more difficult. We previously reported the NMR Molecular Replacement (NMR2) approach that allows the structure of a protein-ligand complex to be determined without requiring the cumbersome task of protein resonance assignment. Herein, we describe the NMR2 approach to determine the binding pose of a small molecule in a weak protein-ligand complex by collecting sparse protein methyl-to-ligand NOEs from a selectively labeled protein sample and an unlabeled ligand. In the selective labeling scheme all methyl containing residues of the protein are protonated in an otherwise deuterated background. This allows measurement of intermolecular NOEs with greater sensitivity using standard NOESY pulse sequences instead of isotope-filtered NMR experiments. This labelling approach is well suited to the NMR2 approach and extends its utility to include larger protein-ligand complexes.

Selective Binding of Small Molecules to Vibrio cholerae DsbA Offers a Starting Point for the Design of Novel Antibacterials.

DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram-negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli. Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X-ray crystal structures reveal that DsbA enzymes share a similar fold, however, the hydrophobic groove adjacent to the active site, which is implicated in substrate binding, is shorter and flatter in the structure of V. cholerae DsbA (VcDsbA) compared to E. coli DsbA (EcDsbA). The flat and largely featureless nature of this hydrophobic groove is challenging for the development of small molecule inhibitors. Using fragment-based screening approaches, we have identified a novel small molecule, based on the benzimidazole scaffold, that binds to the hydrophobic groove of oxidized VcDsbA with a KD of 446±10 µM. The same benzimidazole compound has ∼8-fold selectivity for VcDsbA over EcDsbA and binds to oxidized EcDsbA, with KD >3.5 mM. We generated a model of the benzimidazole complex with VcDsbA using NMR data but were unable to determine the structure of the benzimidazole bound EcDsbA using either NMR or X-ray crystallography. Therefore, a structural basis for the observed selectivity is unclear. To better understand ligand binding to these two enzymes we crystallized each of them in complex with a known ligand, the bile salt sodium taurocholate. The crystal structures show that taurocholate adopts different binding poses in complex with VcDsbA and EcDsbA, and reveal the protein-ligand interactions that stabilize the different modes of binding. This work highlights the capacity of fragment-based drug discovery to identify inhibitors of challenging protein targets. In addition, it provides a starting point for development of more potent and specific VcDsbA inhibitors that act through a novel anti-virulence mechanism.

Structural bioinformatic analysis of DsbA proteins and their pathogenicity associated substrates.

The disulfide bond (DSB) forming system and in particular DsbA, is a key bacterial oxidative folding catalyst. Due to its role in promoting the correct assembly of a wide range of virulence factors required at different stages of the infection process, DsbA is a master virulence rheostat, making it an attractive target for the development of new virulence blockers. Although DSB systems have been extensively studied across different bacterial species, to date, little is known about how DsbA oxidoreductases are able to recognize and interact with such a wide range of substrates. This review summarizes the current knowledge on the DsbA enzymes, with special attention on their interaction with the partner oxidase DsbB and substrates associated with bacterial virulence. The structurally and functionally diverse set of bacterial proteins that rely on DsbA-mediated disulfide bond formation are summarized. Local sequence and secondary structure elements of these substrates are analyzed to identify common elements recognized by DsbA enzymes. This not only provides information on protein folding systems in bacteria but also offers tools for identifying new DsbA substrates and informs current efforts aimed at developing DsbA targeted anti-microbials.

Elaboration of a benzofuran scaffold and evaluation of binding affinity and inhibition of Escherichia coli DsbA: A fragment-based drug design approach to novel antivirulence compounds.

Bacterial thiol-disulfide oxidoreductase DsbA is essential for bacterial virulence factor assembly and has been identified as a viable antivirulence target. Herein, we report a structure-based elaboration of a benzofuran hit that bound to the active site groove of Escherichia coli DsbA. Substituted phenyl groups were installed at the 5- and 6-position of the benzofuran using Suzuki-Miyaura coupling. HSQC NMR titration experiments showed dissociation constants of this series in the high µM to low mM range and X-ray crystallography produced three co-structures, showing binding in the hydrophobic groove, comparable with that of the previously reported benzofurans. The 6-(m-methoxy)phenyl analogue (2b), which showed a promising binding pose, was chosen for elaboration from the C-2 position. The 2,6-disubstituted analogues bound to the hydrophobic region of the binding groove and the C-2 groups extended into the more polar, previously un-probed, region of the binding groove. Biochemical analysis of the 2,6-disubsituted analogues showed they inhibited DsbA oxidation activity in vitro. The results indicate the potential to develop the elaborated benzofuran series into a novel class of antivirulence compounds.

Salmonella enterica BcfH Is a Trimeric Thioredoxin-Like Bifunctional Enzyme with Both Thiol Oxidase and Disulfide Isomerase Activities.

Aims: Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from Salmonella enterica termed bovine colonization factor protein H (BcfH) and defined its role in virulence. Results: In the conserved bovine colonization factor (bcf) fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound α-helix holding the redox active site and a trans-proline instead of the conserved cis-proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to Salmonella fimbrial biogenesis. Innovation and Conclusion: Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity. Antioxid. Redox Signal. 35, 21-39.

Rapid Elaboration of Fragments into Leads by X-ray Crystallographic Screening of Parallel Chemical Libraries (REFiLX).

A bottleneck in fragment-based lead development is the lack of systematic approaches to elaborate the initial fragment hits, which usually bind with low affinity to their target. Herein, we describe an analysis using X-ray crystallography of a diverse library of compounds prepared using microscale parallel synthesis. This approach yielded an 8-fold increase in affinity and detailed structural information for the resulting complex, providing an efficient and broadly applicable approach to early fragment development.

The Fragment-Based Development of a Benzofuran Hit as a New Class of Escherichia coli DsbA Inhibitors.

A fragment-based drug discovery approach was taken to target the thiol-disulfide oxidoreductase enzyme DsbA from Escherichia coli (EcDsbA). This enzyme is critical for the correct folding of virulence factors in many pathogenic Gram-negative bacteria, and small molecule inhibitors can potentially be developed as anti-virulence compounds. Biophysical screening of a library of fragments identified several classes of fragments with affinity to EcDsbA. One hit with high mM affinity, 2-(6-bromobenzofuran-3-yl)acetic acid (6), was chemically elaborated at several positions around the scaffold. X-ray crystal structures of the elaborated analogues showed binding in the hydrophobic binding groove adjacent to the catalytic disulfide bond of EcDsbA. Binding affinity was calculated based on NMR studies and compounds 25 and 28 were identified as the highest affinity binders with dissociation constants (KD) of 326 ± 25 and 341 ± 57 µM respectively. This work suggests the potential to develop benzofuran fragments into a novel class of EcDsbA inhibitors.

The Scs disulfide reductase system cooperates with the metallochaperone CueP in Salmonella copper resistance.

The human pathogen Salmonella enterica serovar Typhimurium (S Typhimurium) contains a complex disulfide bond (Dsb) catalytic machinery. This machinery encompasses multiple Dsb thiol-disulfide oxidoreductases that mediate oxidative protein folding and a less-characterized suppressor of copper sensitivity (scs) gene cluster, associated with increased tolerance to copper. To better understand the function of the Salmonella Scs system, here we characterized two of its key components, the membrane protein ScsB and the periplasmic protein ScsC. Our results revealed that these two proteins form a redox pair in which the electron transfer from the periplasmic domain of ScsB (n-ScsB) to ScsC is thermodynamically driven. We also demonstrate that the Scs reducing pathway remains separate from the Dsb oxidizing pathways and thereby avoids futile redox cycles. Additionally, we provide new insight into the molecular mechanism underlying Scs-mediated copper tolerance in Salmonella We show that both ScsB and ScsC can bind toxic copper(I) with femtomolar affinities and transfer it to the periplasmic copper metallochaperone CueP. Our results indicate that the Salmonella Scs machinery has evolved a dual mode of action, capable of transferring reducing power to the oxidizing periplasm and protecting against copper stress by cooperating with the cue regulon, a major copper resistance mechanism in Salmonella. Overall, these findings expand our understanding of the functional diversity of Dsb-like systems, ranging from those mediating oxidative folding of proteins required for infection to those contributing to defense mechanisms against oxidative stress and copper toxicity, critical traits for niche adaptation and survival.

Unique structural features of a bacterial autotransporter adhesin suggest mechanisms for interaction with host macromolecules.

Autotransporters are the largest family of outer membrane and secreted proteins in Gram-negative bacteria. Most autotransporters are localised to the bacterial surface where they promote colonisation of host epithelial surfaces. Here we present the crystal structure of UpaB, an autotransporter that is known to contribute to uropathogenic E. coli (UPEC) colonisation of the urinary tract. We provide evidence that UpaB can interact with glycosaminoglycans and host fibronectin. Unique modifications to its core ß-helical structure create a groove on one side of the protein for interaction with glycosaminoglycans, while the opposite face can bind fibronectin. Our findings reveal far greater diversity in the autotransporter ß-helix than previously thought, and suggest that this domain can interact with host macromolecules. The relevance of these interactions during infection remains unclear.

Identification of the Binding Site of Apical Membrane Antigen†1 (AMA1) Inhibitors Using a Paramagnetic Probe.

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the 1 H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.

Structural and biochemical insights into the disulfide reductase mechanism of DsbD, an essential enzyme for neisserial pathogens.

The worldwide incidence of neisserial infections, particularly gonococcal infections, is increasingly associated with antibiotic-resistant strains. In particular, extensively drug-resistant Neisseria gonorrhoeae strains that are resistant to third-generation cephalosporins are a major public health concern. There is a pressing clinical need to identify new targets for the development of antibiotics effective against Neisseria-specific processes. In this study, we report that the bacterial disulfide reductase DsbD is highly prevalent and conserved among Neisseria spp. and that this enzyme is essential for survival of N. gonorrhoeae DsbD is a membrane-bound protein that consists of two periplasmic domains, n-DsbD and c-DsbD, which flank the transmembrane domain t-DsbD. In this work, we show that the two functionally essential periplasmic domains of Neisseria DsbD catalyze electron transfer reactions through unidirectional interdomain interactions, from reduced c-DsbD to oxidized n-DsbD, and that this process is not dictated by their redox potentials. Structural characterization of the Neisseria n- and c-DsbD domains in both redox states provides evidence that steric hindrance reduces interactions between the two periplasmic domains when n-DsbD is reduced, thereby preventing a futile redox cycle. Finally, we propose a conserved mechanism of electron transfer for DsbD and define the residues involved in domain-domain recognition. Inhibitors of the interaction of the two DsbD domains have the potential to be developed as anti-neisserial agents.

Inhibition of Diverse DsbA Enzymes in Multi-DsbA Encoding Pathogens.

AIMS: DsbA catalyzes disulfide bond formation in secreted and outer membrane proteins in bacteria. In pathogens, DsbA is a major facilitator of virulence constituting a target for antivirulence antimicrobial development. However, many pathogens encode multiple and diverse DsbA enzymes for virulence factor folding during infection. The aim of this study was to determine whether our recently identified inhibitors of Escherichia coli K-12 DsbA can inhibit the diverse DsbA enzymes found in two important human pathogens and attenuate their virulence. RESULTS: DsbA inhibitors from two chemical classes (phenylthiophene and phenoxyphenyl derivatives) inhibited the virulence of uropathogenic E. coli and Salmonella enterica serovar Typhimurium, encoding two and three diverse DsbA homologues, respectively. Inhibitors blocked the virulence of dsbA null mutants complemented with structurally diverse DsbL and SrgA, suggesting that they were not selective for prototypical DsbA. Structural characterization of DsbA-inhibitor complexes showed that compounds from each class bind in a similar region of the hydrophobic groove adjacent to the Cys30-Pro31-His32-Cys33 (CPHC) active site. Modeling of DsbL- and SrgA-inhibitor interactions showed that these accessory enzymes could accommodate the inhibitors in their different hydrophobic grooves, supporting our in vivo findings. Further, we identified highly conserved residues surrounding the active site for 20 diverse bacterial DsbA enzymes, which could be exploited in developing inhibitors with a broad spectrum of activity. Innovation and Conclusion: We have developed tools to analyze the specificity of DsbA inhibitors in bacterial pathogens encoding multiple DsbA enzymes. This work demonstrates that DsbA inhibitors can be developed to target diverse homologues found in bacteria. Antioxid. Redox Signal. 29, 653-666.

Determination of ligand binding modes in weak protein-ligand complexes using sparse NMR data.

We describe a general approach to determine the binding pose of small molecules in weakly bound protein-ligand complexes by deriving distance constraints between the ligand and methyl groups from all methyl-containing residues of the protein. We demonstrate that using a single sample, which can be prepared without the use of expensive precursors, it is possible to generate high-resolution data rapidly and obtain the resonance assignments of Ile, Leu, Val, Ala and Thr methyl groups using triple resonance scalar correlation data. The same sample may be used to obtain Met εCH3 assignments using NOESY-based methods, although the superior sensitivity of NOESY using [U-13C,15N]-labeled protein makes the use of this second sample more efficient. We describe a structural model for a weakly binding ligand bound to its target protein, DsbA, derived from intermolecular methyl-to-ligand nuclear Overhauser enhancements, and demonstrate that the ability to assign all methyl resonances in the spectrum is essential to derive an accurate model of the structure. Once the methyl assignments have been obtained, this approach provides a rapid means to generate structural models for weakly bound protein-ligand complexes. Such weak complexes are often found at the beginning of programs of fragment based drug design and can be challenging to characterize using X-ray crystallography.

Structure-Activity Studies of ß-Hairpin Peptide Inhibitors of the Plasmodium falciparum AMA1-RON2 Interaction.

The interaction between apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) plays a key role in the invasion of red blood cells by Plasmodium parasites. Disruption of this critical protein-protein interaction represents a promising avenue for antimalarial drug discovery. In this work, we exploited a 13-residue ß-hairpin based on the C-terminal loop of RON2 to probe a conserved binding site on Plasmodium falciparum AMA1. A series of mutations was synthetically engineered into ß-hairpin peptides to establish structure-activity relationships. The best mutations improved the binding affinity of the ß-hairpin peptide by ~7-fold for 3D7 AMA1 and ~14-fold for FVO AMA1. We determined the crystal structures of several ß-hairpin peptides in complex with AMA1 in order to define the structural features and specific interactions that contribute to improved binding affinity. The same mutations in the longer RON2sp2 peptide (residues 2027-2055 of RON2) increased the binding affinity by >30-fold for 3D7 and FVO AMA1, producing KD values of 2.1nM and 0.4nM, respectively. To our knowledge, this is the most potent strain-transcending peptide reported to date and represents a valuable tool to characterize the AMA1-RON2 interaction.

Improved Synthesis of Biotinol-5'-AMP: Implications for Antibacterial Discovery.

An improved synthesis of biotinol-5'-AMP, an acyl-AMP mimic of the natural reaction intermediate of biotin protein ligase (BPL), is reported. This compound was shown to be a pan inhibitor of BPLs from a series of clinically important bacteria, particularly Staphylococcus aureus and Mycobacterium tuberculosis, and kinetic analysis revealed it to be competitive against the substrate biotin. Biotinol-5'-AMP also exhibits antibacterial activity against a panel of clinical isolates of S. aureus and M. tuberculosis with MIC values of 1-8 and 0.5-2.5 µg/mL, respectively, while being devoid of cytotoxicity to human HepG2 cells.

Molecular insights into the interaction between Plasmodium falciparum apical membrane antigen 1 and an invasion-inhibitory peptide.

Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum has been implicated in invasion of the host erythrocyte. It interacts with malarial rhoptry neck (RON) proteins in the moving junction that forms between the host cell and the invading parasite. Agents that block this interaction inhibit invasion and may serve as promising leads for anti-malarial drug development. The invasion-inhibitory peptide R1 binds to a hydrophobic cleft on AMA1, which is an attractive target site for small molecules that block parasite invasion. In this work, truncation and mutational analyses show that Phe5-Phe9, Phe12 and Arg15 in R1 are the most important residues for high affinity binding to AMA1. These residues interact with two well-defined binding hot spots on AMA1. Computational solvent mapping reveals that one of these hot spots is suitable for small molecule targeting. We also confirm that R1 in solution binds to AMA1 with 1:1 stoichiometry and adopts a secondary structure consistent with the major form of R1 observed in the crystal structure of the complex. Our results provide a basis for designing high affinity inhibitors of the AMA1-RON2 interaction.

Ligand-induced conformational change of Plasmodium falciparum AMA1 detected using 19F NMR.

We established an efficient means of probing ligand-induced conformational change in the malaria drug target AMA1 using 19F NMR. AMA1 was labeled with 5-fluorotryptophan (5F-Trp), and the resulting 5F-Trp resonances were assigned by mutagenesis of the native Trp residues. By introducing additional Trp residues at strategic sites within a ligand-responsive loop, we detected distinct conformational consequences when various peptide and small-molecule ligands bound AMA1. Our results demonstrate an increase in flexibility in this loop caused by the native ligand, as inferred from, but not directly observed in, crystal structures. In addition, we found evidence for long-range allosteric changes in AMA1 that are not observed crystallographically. This method will be valuable in ongoing efforts to identify and characterize therapeutically relevant inhibitors of protein-protein interactions involving AMA1 and is generalizable to the study of ligand-induced conformational change in a wide range of other drug targets.