Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor

Abstract

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 ␣/␤ hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-Å 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 ␣/␤ 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.

Signature Motifs Identify an Acinetobacter Cif Virulence Factor with Epoxide Hydrolase Activity

“…Cif has previously been shown to form a dimer in solution (27). To test the oligomeric state of aCif, we calibrated the elution volume of aCif during analytical size-exclusion chromatography relative to a series of mass standards.…”

“…EH Activity-Epoxide hydrolysis activity was first assessed for a panel of candidate epoxides using an adrenochrome reporter end-point assay as described previously (27). Briefly, samples were incubated for 1 h at 37°C in a 100 l volume …”

“…Nevertheless, detailed enzymological studies confirmed that Cif can indeed hydrolyze epoxides (27). Subsequent analysis revealed a series of structural adaptations that enable Cif to retain EH activity despite sequence variations in these active site residues (27,29). Sequence comparisons uncovered similar motifs in the genomes of other opportunistic pathogens, and sequence-structure mapping suggested that these putative EHs are likely to adopt a conformation similar to Cif (29).…”

“…They also include a cis-proline-mediated backbone conformation that helps position an attacking water molecule, and a chargerelay acidic residue C-terminal to the cap domain that helps to activate the water for a second nucleophilic attack, releasing the diol product and regenerating the active site (28). Nevertheless, detailed enzymological studies confirmed that Cif can indeed hydrolyze epoxides (27). Subsequent analysis revealed a series of structural adaptations that enable Cif to retain EH activity despite sequence variations in these active site residues (27,29).…”

“…Sequence and structural homologies indicate that Cif adopts an ␣/␤ hydrolase-fold, a scaffold commonly associated with hydrolytic enzymes (20,27). Based on alignments to other ␣/␤ hydrolase family members, it was originally proposed to act as an epoxide hydrolase (EH).…”