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.

Toward complete rational control over protein structure and function through computational design.

The grand challenge of protein design is a general method for producing a polypeptide with arbitrary functionality, conformation, and biochemical properties. To that end, a wide variety of methods have been developed for the improvement of native proteins, the design of ideal proteins de novo, and the redesign of suboptimal proteins with better-performing substructures. These methods employ informatic comparisons of function-structure-sequence relationships as well as knowledge-based evaluation of protein properties to narrow the immense protein sequence search space down to an enumerable and often manually evaluable set of structures that meet specified criteria. While arbitrary manipulation of protein-protein interfaces and molecular catalysis remains an unsolved problem, and no protein shape or behavior manipulation algorithm is universally applicable, the promising results thus far are a strong indicator that a general approach to the arbitrary manipulation of polypeptides is within reach.

Cytosolic expression, solution structures, and molecular dynamics simulation of genetically encodable disulfide-rich de novo designed peptides.

Disulfide-rich peptides represent an important protein family with broad pharmacological potential. Recent advances in computational methods have made it possible to design new peptides which adopt a stable conformation de novo. Here, we describe a system to produce disulfide-rich de novo peptides using Escherichia coli as the expression host. The advantage of this system is that it enables production of uniformly 13 C- and 15 N-labeled peptides for solution nuclear magnetic resonance (NMR) studies. This expression system was used to isotopically label two previously reported de novo designed peptides, and to determine their solution structures using NMR. The ensemble of NMR structures calculated for both peptides agreed well with the design models, further confirming the accuracy of the design protocol. Collection of NMR data on the peptides under reducing conditions revealed a dependency on disulfide bonds to maintain stability. Furthermore, we performed long-time molecular dynamics (MD) simulations with tempering to assess the stability of two families of de novo designed peptides. Initial designs which exhibited a stable structure during simulations were more likely to adopt a stable structure in vitro, but attempts to utilize this method to redesign unstable peptides to fold into a stable state were unsuccessful. Further work is therefore needed to assess the utility of MD simulation techniques for de novo protein design.

Discovery and engineering of enhanced SUMO protease enzymes.

Small ubiquitin-like modifier (SUMO) is commonly used as a protein fusion domain to facilitate expression and purification of recombinant proteins, and a SUMO-specific protease is then used to remove SUMO from these proteins. Although this protease is highly specific, its limited solubility and stability hamper its utility as an in vitro reagent. Here, we report improved SUMO protease enzymes obtained via two approaches. First, we developed a computational method and used it to re-engineer WT Ulp1 from Saccharomyces cerevisiae to improve protein solubility. Second, we discovered an improved SUMO protease via genomic mining of the thermophilic fungus Chaetomium thermophilum, as proteins from thermophilic organisms are commonly employed as reagent enzymes. Following expression in Escherichia coli, we found that these re-engineered enzymes can be more thermostable and up to 12 times more soluble, all while retaining WT-or-better levels of SUMO protease activity. The computational method we developed to design solubility-enhancing substitutions is based on the RosettaScripts application for the macromolecular modeling suite Rosetta, and it is broadly applicable for the improvement of solution properties of other proteins. Moreover, we determined the X-ray crystal structure of a SUMO protease from C. thermophilum to 1.44 Å resolution. This structure revealed that this enzyme exhibits structural and functional conservation with the S. cerevisiae SUMO protease, despite exhibiting only 28% sequence identity. In summary, by re-engineering the Ulp1 protease and discovering a SUMO protease from C. thermophilum, we have obtained proteases that are more soluble, more thermostable, and more efficient than the current commercially available Ulp1 enzyme.

Screening, large-scale production and structure-based classification of cystine-dense peptides.

Peptides folded through interwoven disulfides display extreme biochemical properties and unique medicinal potential. However, their exploitation has been hampered by the limited amounts isolatable from natural sources and the expense of chemical synthesis. We developed reliable biological methods for high-throughput expression, screening and large-scale production of these peptides: 46 were successfully produced in multimilligram quantities, and >600 more were deemed expressible through stringent screening criteria. Many showed extreme resistance to temperature, proteolysis and/or reduction, and all displayed inhibitory activity against at least 1 of 20 ion channels tested, thus confirming their biological functionality. Crystal structures of 12 confirmed proper cystine topology and the utility of crystallography to study these molecules but also highlighted the need for rational classification. Previous categorization attempts have focused on limited subsets featuring distinct motifs. Here we present a global definition, classification and analysis of >700 structures of cystine-dense peptides, providing a unifying framework for these molecules.

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.

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.

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.

Accurate de novo design of hyperstable constrained peptides.

Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes that have evolved to fit precisely into binding pockets on their targets. Such peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small-molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for accurate de novo design of conformationally restricted peptides, and the use of these methods to design 18-47 residue, disulfide-crosslinked peptides, a subset of which are heterochiral and/or N-C backbone-cyclized. Both genetically encodable and non-canonical peptides are exceptionally stable to thermal and chemical denaturation, and 12 experimentally determined X-ray and NMR structures are nearly identical to the computational design models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs.

Inhibiting an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa Protects CFTR.

Opportunistic pathogens exploit diverse strategies to sabotage host defenses. Pseudomonas aeruginosa secretes the CFTR inhibitory factor Cif and thus triggers loss of CFTR, an ion channel required for airway mucociliary defense. However, the mechanism of action of Cif has remained unclear. It catalyzes epoxide hydrolysis, but there is no known role for natural epoxides in CFTR regulation. It was demonstrated that the hydrolase activity of Cif is strictly required for its effects on CFTR. A small-molecule inhibitor that protects this key component of the mucociliary defense system was also uncovered. These results provide a basis for targeting the distinctive virulence chemistry of Cif and suggest an unanticipated role of physiological epoxides in intracellular protein trafficking.

Signature motifs identify an Acinetobacter Cif virulence factor with epoxide hydrolase activity.

Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.

Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for ß-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

Epoxide-mediated CifR repression of cif gene expression utilizes two binding sites in Pseudomonas aeruginosa.

Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB, the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the -10 and +1 sites of both the cifR and morB regulatory region and the start of transcription, respectively. In addition, we found that CifR binds to each repression site with differing affinity. Mutagenesis of these binding sites resulted in a loss of DNA binding in vitro, and mutation of one of these sites in vivo resulted in an increase in transcription of both the cif and cifR genes. We characterized cif and cifR gene expression in sputum and found that, whereas cif gene expression varied relative to an in vitro coculture control, cifR gene expression was consistently higher. Analysis of a longitudinal sample of CF isolates from nine patients revealed that Cif protein was expressed over time, although variably, and these changes could not be linked to mutations in the cifR gene or the promoters of these genes. Finally, we tested CifR responsiveness to other epoxides and showed that CifR can respond to multiple epoxides to various degrees.

Pseudomonas aeruginosa Cif defines a distinct class of α/ß epoxide hydrolases utilizing a His/Tyr ring-opening pair.

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that secretes a multitude of virulence factors during the course of infection. Among these is Cif, an epoxide hydrolase (EH) that reduces the functional localization of the cystic fibrosis transmembrane conductance regulator in epithelial cells. In addition to being the first reported EH virulence factor, Cif possesses unique sequence deviations from canonical EH motifs. Foremost among these is the substitution of a histidine for the first epoxide ring-opening tyrosine in the active site. To test the functional equivalence of Tyr and His side chains at this position, we have generated the mutant Cif-H177Y. Structural analysis confirms that both the WT His and mutant Tyr side chains can be accommodated without large-scale conformational changes. However, the Tyr mutant is functionally inactive. Based on a detailed analysis of the structure of the Tyr mutant, it appears that Cif's main-chain conformation imposes a functional requirement for a His at this position. Comparison with canonical EH structures reveals additional conformational differences, which are coupled to divergent sequence characteristics. When used to probe the genomes of other opportunistic pathogens, these sequence-structure criteria uncover candidate sequences that appear to form a distinct subfamily of Cif-like epoxide hydrolases characterized by a conserved His/Tyr ring-opening pair.

Crystal structure of the cystic fibrosis transmembrane conductance regulator inhibitory factor Cif reveals novel active-site features of an epoxide hydrolase virulence factor.

Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other alpha/beta hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-A resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of alpha/beta hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.

Purification, crystallization and preliminary X-ray diffraction analysis of Cif, a virulence factor secreted by Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa secretes a protein that triggers the accelerated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells. This protein, which is known as the CFTR inhibitory factor (Cif), acts as a virulence factor and may facilitate airway colonization by P. aeruginosa. Based on sequence similarity Cif appears to be an epoxide hydrolase (EH), but it lacks several of the conserved features found in the active sites of canonical members of the EH family. Here, the crystallization of purified recombinant Cif by vapor diffusion is reported. The crystals formed in space group C2, with unit-cell parameters a = 167.4, b = 83.6, c = 88.3 A, beta = 100.6 degrees . The crystals diffracted to 2.39 A resolution on a rotating-anode source. Based on the calculated Matthews coefficient (2.2 A(3) Da(-1)), it appears that the asymmetric unit contains four molecules.