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 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.

FIG. 1.

Epoxide hydrolase activity. The EH class of enzymes is responsible for the catalytic addition of a water molecule to an epoxide ring, creating a vicinal diol.

FIG. 2.

Crystal structure of Cif. (A) 2F_o-F_c map of the final refined electron density of Cif-WT, contoured to 1σ; the HGFG motif (residues 61 to 64) is colored green. A density mask with a 1.8-Å radius has been applied to prevent the display of electron density from neighboring residues. (B) Ribbon diagram depicting the superposition of ArEH and Cif-WT by DaliLite v.3 (28). The Cif core domain is colored in gray, and the cap domain is in yellow; ArEH is colored with a green core and a blue cap domain.

FIG. 3.

Sequence and structural similarity to ArEH. The sequences of Cif and ArEH were aligned, and the secondary structure was determined using DaliLite v.3 (28). Residues 138 to 148, which are shown in lowercase letters, are absent from the crystal structure of ArEH (PDB ID 1EHY) and were aligned using ClustalW (51). Arrows indicate β strands, and the relative orientation within a sheet is indicated by the direction of the arrow. The cap domain of each protein is underlined. The secondary structure elements of ArEH are named according to Nardini et al. (43). Amino acids are colored as follows: small side chains are orange (Gly and Ala), Pro is brown, Cys is maroon, polar side chains are gray (Ser, Thr, Asn, and Gln), acidic side chains are red (Asp and Glu), basic side chains are blue (His, Arg, and Lys), nonpolar side chains are green (Ile, Leu, Met, and Val), and aromatic side chains are cyan (Phe, Tyr, and Trp). Symbols: ‡, HGFG motif; *, active-site residues, including His177; †, residue His269 of Cif.

FIG. 4.

Cif forms a homodimer in solution. (A) The sedimentation coefficient concentration distribution c(S) was determined for Cif using velocity sedimentation analysis. The single peak is shown at 4.3S. (B) SEC of Cif-WT reveals a single peak at V_e = 15.76 ml. Arrows indicate the elution volumes of standard proteins used to calibrate the R_s of Cif. Void volume (V₀), 8.90 ml; aldolase (ald), 13.04 ml; ovalbumin (ova), 15.72 ml; chymotrypsinogen A (chy), 17.54 ml; RNase A (RNa), 18.54 ml; and total volume (V_t), 21.93 ml.

FIG. 5.

Substrate selectivity of Cif. (A) Buffer-subtracted hydrolysis of 50 μM radiolabeled canonical epoxide hydrolase substrates by 1 μM Cif-WT. Cif exhibited significant hydrolytic activity only for CSO. (B) Ribbon diagram of the Cif homodimer, as seen down the 2-fold axis. The side chains of active-site residues Asp129, Glu153, His177, Try239, and His297 and the HGFG motif ribbon are shown in blue. The calculated tunnel from the solvent to Asp129 for each monomer is shown in red.

FIG. 6.

Cif active site. (A) Ribbon diagram of Cif-WT. The cap domain, consisting of residues 155 to 242, is colored yellow, and the core is in gray. The side chains of active-site residues are modeled as sticks, and the main chain of the HGFG motif is shown with the carbons colored light blue. (B) A detailed view of the active site. Hydrogen bonds are shown as dotted lines and the main chain as a C_α trace. The carboxylate of Glu153 participates in three hydrogen bonds. One is accepted from His297 and two from the protein backbone via the amide nitrogens of Gly266 and Met272. Asp129 is positioned by hydrogen bonds donated from the amide nitrogens of neighboring residue Ile130 and of Phe63 of the HGFG motif. W1 is coordinated by hydrogen bonds to Tyr239 and His177. W2 donates hydrogen bonds to the carbonyl oxygen of Phe63 and the imidazole of His297.

FIG. 7.

Reassessment of Cif-H269A EH and CFTR inhibitory activity. (A) C_α traces for Cif-WT (green) and Cif-H269A (blue) are shown following superposition, which yielded an rmsd of 0.08 Å. The side chains of residue 269 for both structures are shown in red. (B) Hydrolysis of 10 mM EBH by 20 μM Cif-WT or Cif-H269A. (C) Hydrolysis of radiolabeled CSO (50 μM) by Cif-WT and Cif-H269A (1 μM). (D) Cif-WT decreases the apical membrane abundance of CFTR, while the H269A mutation abrogates this effect. Fifty μg of either Cif-WT or Cif-H269A was added to the apical surface of CFBE WT-CFTR cells and incubated for 60 min, followed by Western blot analysis to determine the apical membrane abundance of CFTR. Samples are scaled in comparison to the buffer control, which was normalized to 100%.