Molecular basis for the transcriptional regulation of an epoxide-based virulence circuit in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa infects cystic fibrosis (CF) patient airways and produces a virulence factor Cif that is associated with worse outcomes. Cif is an epoxide hydrolase that reduces cell-surface abundance of the cystic fibrosis transmembrane conductance regulator (CFTR) and sabotages pro-resolving signals. Its expression is regulated by a divergently transcribed TetR family transcriptional repressor. CifR represents the first reported epoxide-sensing bacterial transcriptional regulator, but neither its interaction with cognate operator sequences nor the mechanism of activation has been investigated. Using biochemical and structural approaches, we uncovered the molecular mechanisms controlling this complex virulence operon. We present here the first molecular structures of CifR alone and in complex with operator DNA, resolved in a single crystal lattice. Significant conformational changes between these two structures suggest how CifR regulates the expression of the virulence gene cif. Interactions between the N-terminal extension of CifR with the DNA minor groove of the operator play a significant role in the operator recognition of CifR. We also determined that cysteine residue Cys107 is critical for epoxide sensing and DNA release. These results offer new insights into the stereochemical regulation of an epoxide-based virulence circuit in a critically important clinical pathogen.

Additive energetic contributions of multiple peptide positions determine the relative promiscuity of viral and human sequences for PDZ domain targets.

Protein-protein interactions that involve recognition of short peptides are critical in cellular processes. Protein-peptide interaction surface areas are relatively small and shallow, and there are often overlapping specificities in families of peptide-binding domains. Therefore, dissecting selectivity determinants can be challenging. PDZ domains are a family of peptide-binding domains located in several intracellular signaling and trafficking pathways. These domains are also directly targeted by pathogens, and a hallmark of many oncogenic viral proteins is a PDZ-binding motif. However, amidst sequences that target PDZ domains, there is a wide spectrum in relative promiscuity. For example, the viral HPV16 E6 oncoprotein recognizes over double the number of PDZ domain-containing proteins as the cystic fibrosis transmembrane conductance regulator (CFTR) in the cell, despite similar PDZ targeting-sequences and identical motif residues. Here, we determine binding affinities for PDZ domains known to bind either HPV16 E6 alone or both CFTR and HPV16 E6, using peptides matching WT and hybrid sequences. We also use energy minimization to model PDZ-peptide complexes and use sequence analyses to investigate this difference. We find that while the majority of single mutations had marginal effects on overall affinity, the additive effect on the free energy of binding accurately describes the selectivity observed. Taken together, our results describe how complex and differing PDZ interactomes can be programmed in the cell.

Additive energetic contributions of multiple peptide positions determine the relative promiscuity of viral and human sequences for PDZ domain targets.

Protein-protein interactions that include recognition of short sequences of amino acids, or peptides, are critical in cellular processes. Protein-peptide interaction surface areas are relatively small and shallow, and there are often overlapping specificities in families of peptide-binding domains. Therefore, dissecting selectivity determinants can be challenging. PDZ domains are an example of a peptide-binding domain located in several intracellular signaling and trafficking pathways, which form interactions critical for the regulation of receptor endocytic trafficking, tight junction formation, organization of supramolecular complexes in neurons, and other biological systems. These domains are also directly targeted by pathogens, and a hallmark of many oncogenic viral proteins is a PDZ-binding motif. However, amidst sequences that target PDZ domains, there is a wide spectrum in relative promiscuity. For example, the viral HPV16 E6 oncoprotein recognizes over double the number of PDZ domain-containing proteins as the cystic fibrosis transmembrane conductance regulator (CFTR) in the cell, despite similar PDZ targeting-sequences and identical motif residues. Here, we determine binding affinities for PDZ domains known to bind either HPV16 E6 alone or both CFTR and HPV16 E6, using peptides matching WT and hybrid sequences. We also use energy minimization to model PDZ-peptide complexes and use sequence analyses to investigate this difference. We find that while the majority of single mutations had a marginal effect on overall affinity, the additive effect on the free energy of binding accurately describes the selectivity observed. Taken together, our results describe how complex and differing PDZ interactomes can be programmed in the cell.

Tryptophan mutations in G3BP1 tune the stability of a cellular signaling hub by weakening transient interactions with Caprin1 and USP10.

Intrinsically disordered proteins (IDPs) often coordinate transient interactions with multiple proteins to mediate complex signals within large protein networks. Among these, the IDP hub protein G3BP1 can form complexes with cytoplasmic phosphoprotein Caprin1 and ubiquitin peptidase USP10; the resulting control of USP10 activity contributes to a pathogenic virulence system that targets endocytic recycling of the ion channel CFTR. However, while the identities of protein interactors are known for many IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood. Here, we describe in vitro analysis of these G3BP1 affinities and show tryptophan substitutions of specific G3BP1 residues reduce its affinity for both USP10 and Caprin1. We show that these same mutations reduce the stability of complexes between the full-length proteins, suggesting that copurification can serve as a surrogate measure of interaction strength. The crystal structure of G3BP1 TripleW (F15W/F33W/F124W) mutant reveals a clear reorientation of the side chain of W33, creating a steric clash with USP10 and Caprin1. Furthermore, an amino-acid scan of USP10 and Caprin1 peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific substitutions with the potential to create higher affinity peptides. Taken together, these data show that small changes in component binding affinities can have significant effects on the composition of cellular interaction hubs. These specific protein mutations can be harnessed to manipulate complex protein networks, informing future investigations into roles of these networks in cellular processes.

Model Systems to Study the Chronic, Polymicrobial Infections in Cystic Fibrosis: Current Approaches and Exploring Future Directions.

A recent workshop titled "Developing Models to Study Polymicrobial Infections," sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 35+ investigators over two virtual sessions. Here, we present the findings of this workshop, summarize some of the challenges involved with developing such models, and suggest three frameworks to tackle this complex problem. The frameworks proposed here, we believe, could be generally useful in developing new model systems for other infectious diseases. Developing and validating new approaches to study the complex polymicrobial communities in the CF airway could open windows to new therapeutics to treat these recalcitrant infections, as well as uncovering organizing principles applicable to chronic polymicrobial infections more generally.

Biochemical and structural characterization of two cif-like epoxide hydrolases from Burkholderia cenocepacia.

Epoxide hydrolases catalyze the conversion of epoxides to vicinal diols in a range of cellular processes such as signaling, detoxification, and virulence. These enzymes typically utilize a pair of tyrosine residues to orient the substrate epoxide ring in the active site and stabilize the hydrolysis intermediate. A new subclass of epoxide hydrolases that utilize a histidine in place of one of the tyrosines was established with the discovery of the CFTR Inhibitory Factor (Cif) from Pseudomonas aeruginosa. Although the presence of such Cif-like epoxide hydrolases was predicted in other opportunistic pathogens based on sequence analyses, only Cif and its homolog aCif from Acinetobacter nosocomialis have been characterized. Here we report the biochemical and structural characteristics of Cfl1 and Cfl2, two Cif-like epoxide hydrolases from Burkholderia cenocepacia. Cfl1 is able to hydrolyze xenobiotic as well as biological epoxides that might be encountered in the environment or during infection. In contrast, Cfl2 shows very low activity against a diverse set of epoxides. The crystal structures of the two proteins reveal quaternary structures that build on the well-known dimeric assembly of the α/ß hydrolase domain, but broaden our understanding of the structural diversity encoded in novel oligomer interfaces. Analysis of the interfaces reveals both similarities and key differences in sequence conservation between the two assemblies, and between the canonical dimer and the novel oligomer interfaces of each assembly. Finally, we discuss the effects of these higher-order assemblies on the intra-monomer flexibility of Cfl1 and Cfl2 and their possible roles in regulating enzymatic activity.

Specificity in PDZ-peptide interaction networks: Computational analysis and review.

Globular PDZ domains typically serve as protein-protein interaction modules that regulate a wide variety of cellular functions via recognition of short linear motifs (SLiMs). Often, PDZ mediated-interactions are essential components of macromolecular complexes, and disruption affects the entire scaffold. Due to their roles as linchpins in trafficking and signaling pathways, PDZ domains are attractive targets: both for controlling viral pathogens, which bind PDZ domains and hijack cellular machinery, as well as for developing therapies to combat human disease. However, successful therapeutic interventions that avoid off-target effects are a challenge, because each PDZ domain interacts with a number of cellular targets, and specific binding preferences can be difficult to decipher. Over twenty-five years of research has produced a wealth of data on the stereochemical preferences of individual PDZ proteins and their binding partners. Currently the field lacks a central repository for this information. Here, we provide this important resource and provide a manually curated, comprehensive list of the 271 human PDZ domains. We use individual domain, as well as recent genomic and proteomic, data in order to gain a holistic view of PDZ domains and interaction networks, arguing this knowledge is critical to optimize targeting selectivity and to benefit human health.

Computational Analysis of Energy Landscapes Reveals Dynamic Features That Contribute to Binding of Inhibitors to CFTR-Associated Ligand.

The CFTR-associated ligand PDZ domain (CALP) binds to the cystic fibrosis transmembrane conductance regulator (CFTR) and mediates lysosomal degradation of mature CFTR. Inhibition of this interaction has been explored as a therapeutic avenue for cystic fibrosis. Previously, we reported the ensemble-based computational design of a novel peptide inhibitor of CALP, which resulted in the most binding-efficient inhibitor to date. This inhibitor, kCAL01, was designed using osprey and evinced significant biological activity in in vitro cell-based assays. Here, we report a crystal structure of kCAL01 bound to CALP and compare structural features against iCAL36, a previously developed inhibitor of CALP. We compute side-chain energy landscapes for each structure to not only enable approximation of binding thermodynamics but also reveal ensemble features that contribute to the comparatively efficient binding of kCAL01. Finally, we compare the previously reported design ensemble for kCAL01 vs the new crystal structure and show that, despite small differences between the design model and crystal structure, significant biophysical features that enhance inhibitor binding are captured in the design ensemble. This suggests not only that ensemble-based design captured thermodynamically significant features observed in vitro, but also that a design eschewing ensembles would miss the kCAL01 sequence entirely.

Nanobody-based binding assay for the discovery of potent inhibitors of CFTR inhibitory factor (Cif).

Lead identification and optimization are essential steps in the development of a new drug. It requires cost-effective, selective and sensitive chemical tools. Here, we report a novel method using nanobodies that allows the efficient screening for potent ligands. The method is illustrated with the cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), a virulence factor secreted by the opportunistic pathogen Pseudomonas aeruginosa. 18 nanobodies selective to Cif were isolated by bio-panning from nanobody-phage library constructed from immunized llama. 8 out of 18 nanobodies were identified as potent inhibitors of Cif enzymatic activity with IC50s in the range of 0.3-6.4 µM. A nanobody VHH219 showed high affinity (KD = 0.08 nM) to Cif and the highest inhibitory potency, IC50 = 0.3 µM. A displacement sandwich ELISA (dsELISA) with VHH219 was then developed for classification of synthetic small molecule inhibitors according their inhibitory potency. The developed assay allowed identification of new inhibitor with highest potency reported so far (0.16 ±â€¯0.02 µM). The results from dsELISA assay correlates strongly with a conventional fluorogenic assay (R = 0.9998) in predicting the inhibitory potency of the tested compounds. However, the novel dsELISA is an order of magnitude more sensitive and allows the identification and ranking of potent inhibitors missed by the classic fluorogenic assay method. These data were supported with Octet biolayer interferometry measurements. The novel method described herein relies solely on the binding properties of the specific neutralizing nanobody, and thus is applicable to any pharmacological target for which such a nanobody can be found, independent of any requirement for catalytic activity.

Cysteine modifiers suggest an allosteric inhibitory site on the CAL PDZ domain.

Protein-protein interactions have become attractive targets for both experimental and therapeutic interventions. The PSD-95/Dlg1/ZO-1 (PDZ) domain is found in a large family of eukaryotic scaffold proteins that plays important roles in intracellular trafficking and localization of many target proteins. Here, we seek inhibitors of the PDZ protein that facilitates post-endocytic degradation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR): the CFTR-associated ligand (CAL). We develop and validate biochemical screens and identify methyl-3,4-dephostatin (MD) and its analog ethyl-3,4-dephostatin (ED) as CAL PDZ inhibitors. Depending on conditions, MD can bind either covalently or non-covalently. Crystallographic and NMR data confirm that MD attacks a pocket at a site distinct from the canonical peptide-binding groove, and suggests an allosteric connection between target residue Cys319 and the conserved Leu291 in the GLGI motif. MD and ED thus appear to represent the first examples of small-molecule allosteric regulation of PDZ:peptide affinity. Their mechanism of action may exploit the known conformational plasticity of the PDZ domains and suggests that allosteric modulation may represent a strategy for targeting of this family of protein-protein binding modules.

An epoxide hydrolase secreted by Pseudomonas aeruginosa decreases mucociliary transport and hinders bacterial clearance from the lung.

The opportunistic pathogen Pseudomonas aeruginosa colonizes the lungs of susceptible individuals by deploying virulence factors targeting host defenses. The secreted factor Cif (cystic fibrosis transmembrane conductance regulator inhibitory factor) dysregulates the endocytic recycling of CFTR and thus reduces CFTR abundance in host epithelial membranes. We have postulated that the decrease in ion secretion mediated by Cif would slow mucociliary transport and decrease bacterial clearance from the lungs. To test this hypothesis, we explored the effects of Cif in cultured epithelia and in the lungs of mice. We developed a strategy to interpret the "hurricane-like" motions observed in reconstituted cultures and identified a Cif-mediated decrease in the velocity of mucus transport in vitro. Presence of Cif also increased the number of bacteria recovered at two time points in an acute mouse model of pneumonia caused by P. aeruginosa. Furthermore, recent work has demonstrated an inverse correlation between the airway concentrations of Cif and 15-epi-lipoxin A4, a proresolving lipid mediator important in host defense and the resolution of pathogen-initiated inflammation. Here, we observe elevated levels of 15-epi-lipoxin A4 in the lungs of mice infected with a strain of P. aeruginosa that expresses only an inactive form of cif compared with those mice infected with wild-type P. aeruginosa. Together these data support the inclusion of Cif on the list of virulence factors that assist P. aeruginosa in colonizing and damaging the airways of compromised patients. Furthermore, this study establishes techniques that enable our groups to explore the underlying mechanisms of Cif effects during respiratory infection.

Optimization of the process of inverted peptides (PIPEPLUS) to screen PDZ domain ligands.

PDZ domains play crucial roles in cell signaling processes and are therefore attractive targets for the development of therapeutic inhibitors. In many cases, C-terminal peptides are the physiological binding partners of PDZ domains. To identify both native ligands and potential inhibitors we have screened arrays synthesized by the process of inverted peptides (PIPE), a variant of SPOT synthesis that generates peptides with free C-termini. Here, we present the development of a new functionalized cellulose membrane as solid support along with the optimized PIPEPLUS technology. Improved resolution and accuracy of the synthesis were shown with peptide arrays containing both natural and non-natural amino acids. These new screening possibilities will advance the development of active, selective and metabolically stable PDZ interactors.

Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase.

Pseudomonas aeruginosa secretes an epoxide hydrolase with catalytic activity that triggers degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and perturbs other host defense networks. Targets of this CFTR inhibitory factor (Cif) are largely unknown, but include an epoxy-fatty acid. In this class of signaling molecules, chirality can be an important determinant of physiological output and potency. Here we explore the active-site chemistry of this two-step α/ß-hydrolase and its implications for an emerging class of virulence enzymes. In combination with hydrolysis data, crystal structures of 15 trapped hydroxyalkyl-enzyme intermediates reveal the stereochemical basis of Cif's substrate specificity, as well as its regioisomeric and enantiomeric preferences. The structures also reveal distinct sets of conformational changes that enable the active site to expand dramatically in two directions, accommodating a surprising array of potential physiological epoxide targets. These new substrates may contribute to Cif's diverse effects in vivo, and thus to the success of P. aeruginosa and other pathogens during infection.

The CFTR trafficking mutation F508del inhibits the constitutive activity of SLC26A9.

Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.

The cif Virulence Factor Gene Is Present in Isolates From Patients With Pseudomonas aeruginosa Keratitis.

PURPOSE: To determine whether the cif gene is present in pathogenic Pseudomonas aeruginosa isolates from patients with bacterial keratitis at Aravind Eye Hospital, a referral eye care center in southern India, and from corresponding environmental isolates. METHODS: Polymerase chain reaction amplification was performed on strains of P. aeruginosa isolated from ocular infections and environmental soil samples were collected from the area surrounding Aravind Eye Hospital. DNA sequencing of 16S ribosomal DNA amplicons was performed to verify strain identity. RESULTS: We determined that 45 of 48 patient isolates carry a genomic copy of cif. Analysis of a catalog of environmental strains previously isolated from the surrounding area revealed that only 4 of 10 P. aeruginosa strains and 1 of 14 strains of related species carry the cif gene. CONCLUSIONS: This is the first study to show that P. aeruginosa strains with ocular pathogenicity carry the cif gene and that the presence of this gene may be enriched over its prevalence in the environment. Taken together, these results suggest a potential role for Cif in acute bacterial keratitis.

Pseudomonas aeruginosa sabotages the generation of host proresolving lipid mediators.

Recurrent Pseudomonas aeruginosa infections coupled with robust, damaging neutrophilic inflammation characterize the chronic lung disease cystic fibrosis (CF). The proresolving lipid mediator, 15-epi lipoxin A4 (15-epi LXA4), plays a critical role in limiting neutrophil activation and tissue inflammation, thus promoting the return to tissue homeostasis. Here, we show that a secreted P. aeruginosa epoxide hydrolase, cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), can disrupt 15-epi LXA4 transcellular biosynthesis and function. In the airway, 15-epi LXA4 production is stimulated by the epithelial-derived eicosanoid 14,15-epoxyeicosatrienoic acid (14,15-EET). Cif sabotages the production of 15-epi LXA4 by rapidly hydrolyzing 14,15-EET into its cognate diol, eliminating a proresolving signal that potently suppresses IL-8-driven neutrophil transepithelial migration in vitro. Retrospective analyses of samples from patients with CF supported the translational relevance of these preclinical findings. Elevated levels of Cif in bronchoalveolar lavage fluid were correlated with lower levels of 15-epi LXA4, increased IL-8 concentrations, and impaired lung function. Together, these findings provide structural, biochemical, and immunological evidence that the bacterial epoxide hydrolase Cif disrupts resolution pathways during bacterial lung infections. The data also suggest that Cif contributes to sustained pulmonary inflammation and associated loss of lung function in patients with CF.

A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain.

A method for development of murine Fab fragments towards extracellular domains of a surface receptor is presented. The GluA4 ionotropic glutamate receptor is used as a model system. Recombinant GluA4 ectodomain comprising both the N-terminal domain (NTD) and the ligand-binding domain (LBD) in one molecule was used for immunization. A Fab-phage library was constructed and a parallel panning approach enabled selection of murine Fab fragments towards either intact ectodomain or the isolated LBD of the GluA4 receptor. One LBD-Fab (FabL9) showed exclusive selectivity for the GluA4 LBD, over a panel of LBDs from GluA2, GluK1, GluK2 and GluD2. Soluble FabL9 was produced in amounts suitable for characterization. Competitive ELISA and rat-brain immunoprecipitation experiments confirmed that the FabL9 epitope is conserved in the LBD and in the intact native receptor. By an alignment of GluA2 and GluA4, the likely binding epitope for FabL9 was predicted. This study demonstrates a simple approach for development of antibody fragments towards specific sub-domains of a large ligand-gated ion channel, and this method could be utilized for all multi-domain surface receptors where antibody domain-selectivity may be desirable. Furthermore, we present for the first time a GluA4 subtype-specific murine Fab fragment targeting the LBD of the receptor.

Rational Design of Potent and Selective Inhibitors of an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa.

The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. On the basis of these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif and have the potential for clinical application.