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

Abstract

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.

Keywords: Epoxide-based virulence circuit; Protein-DNA recognition; Pseudomonas aeruginosa; TetR family transcriptional regulators (TFRs); X-ray crystallography.

Figure 1.

The cif-cifR operon and CifR protein solubility. (A) Top row: The cif-cifR operon. The arrows show the four open reading frames in the operon. The two rectangles in grey represent the two operator sites for CifR binding (Middle row; blue). Bottom row: Binding to an epoxide (schematic drawing) releases CifR from the DNA. Cif is shown as a gold oval doublet which converts the epoxide ring to a vicinal diol (schematic drawing). (B-D) Coomassie-stained SDS-PAGE gels for CifR constructs. Rosetta 2 (DE3) cells were transformed with plasmids expressing decahistidine-tagged WT (B), CifR_T (C), and CifR_TR (D) constructs, grown in LB medium, and induced with 0.5 mM IPTG overnight at 16 °C. Lanes are marked “L” for whole-cell lysate or “S” for supernatant clarified by centrifugation at 40,000 rpm for 1 hr in a 45 Ti rotor. The positions of M_r standards are shown for each gel (adjacent lines) and the corresponding M_r values shown in (E). The short black lines on the side of the gel indicates protein standards of size 76 kDa, 52 kDa, 38 kDa, 31 kDa, 24 kDa, 17 kDa and 12 kDa from top to bottom. The black arrows point to the expected gel migration position for CifR monomer according to the protein standards. The grey arrow points to the expected gel migration position for CifR dimer. (E) SDS-PAGE gel of purified CifR_TR (in the absence and presence of reducing agent) following cleavage of the decahistidine tag.

Figure 2.

DNA screening for CifR_TR:DNA complex formation and crystallization. (A) Sequences of the DNA oligonucleotides tested for optimizing the CifR_TR:DNA complex formation. Varying sequence lengths of the cifR-proximal operator DNA (2^nd row) were assessed for CifR_TR:DNA complex formation and co-crystallization. The sequence highlighted in grey represents the minimal length of DNA (23bp) which could bind to CifR in this study. Cohesive bases are highlighted in red. Sequence alignment of the cifR-proximal and the morB-proximal operator (Top row) is shown. The two operators present in an inverted orientation in the genome of P. aeruginosa. The orientation of the strands labeled with 5’ to 3’ correspond to the top strand in Figure 1A. (B) EMSAs of CifR_TR with each of the oligonucleotides listed in (A). The concentration of DNA in the assay was 2 μM. “P:D ratio” is the molar ratio of CifR monomer to DNA. “P:D” and “DNA” indicate the migration positions of protein:DNA complex and free DNA, respectively. This shorthand is used throughout the article. (C) Representative crystallization screening results for each complex assayed in (B). Each picture shows the crystal morphology with the best diffraction, or the most common result observed during the screening process.

Figure 3.

Lattice packing of the CifR_TR:dsDNA_26L complex and apo-CifR_TR crystal. (A) Lattice formation by DNA- and protein-protein interactions. DNA duplexes (orange/grey) align end-to-end to form extended helices, with each DNA molecule bound by one CifR_TR dimer (green ribbons). The DNA duplexes in the center are shown in grey to distinguish the DNA-DNA junctions. A 90° rotation about the indicated axis reveals the protein-protein contacts that join neighboring DNA helical extensions in the lattice (right). (B) Apo-CifR_TR is caged into the bulk solvent channel of the lattice formed by CifR_TR:dsDNA_26L. The panel on the top left shows the bulk solvent channels formed by the CifR_TR:dsDNA_26L complex. The inset on the top right shows a closeup view of the apo-CifR_TR dimers (blue ribbons), which are omitted in the parent panel. The inset on the bottom right shows the 90° rotation view of the inset on the top right. The inset on the bottom left shows the close up view of lattice contacts between the apo-Cif_TR and CifR_TR:dsDNA_26L complex. Residues with polar contacts are shown as sticks.

Figure 4.

Structural analysis of the CifR_TR-dsDNA_26L complex. (A) Overall structure of the CifR_TR:dsDNA_26L complex. CifR_TR contacts the DNA duplex (orange) through the DNA-binding domain (DBD, green ribbon), while the ligand-binding domain (LBD, pale cyan ribbon) is distal to the protein-DNA interface. The nine helices that make up the bulk of the CifR_TR structure are labeled for one protomer. The distance between the two Pro45 residues in CifR dimer is shown. (B) Protein:DNA contact interface at the minor groove. Arg6 (green stick model) inserts into the DNA minor groove and forms hydrogen bonds (grey dashes) with several DNA bases (dT4, dT5, dT6, dA23’, orange sticks) and a water molecule (blue sphere). Pro44 and Pro45 are shown as green sticks. (C) Protein:DNA contact interface at the major groove. Leu33, Tyr48, Lys54 (green sticks) form hydrogen bonds (grey dashes) with backbone atoms of dG16’, dA17’ (orange sticks). Pro44 and Pro45 (green sticks) insert into the major groove but do not form hydrogen bonds. (D) Two-dimensional schematic of CifR-DNA interactions. The central dotted black line indicates the two-fold symmetry axis of the CifR dimer. Lines indicate interactions with DNA bases (red) or with the DNA backbone or a water molecule (black). (E) Functional analysis of protein-DNA contacts by EMSA. The DNA concentration was 0.5 μM. Protein concentrations tested were 0, 0.1, 0.2, 0.4, 0.8, 1, 1.6, 2, 4 and 10 μM for the nine titrations (panels 1–3), and 1, 2, and 5 μM for the three titrations (panel 4). Duplex dsDNA_26L^T4G has mutations at T4 and A23. Duplex dsDNA_26L^4-mix has mutations at C15T, A16C, C17T and T18G (see Table S1). R6A: CifR_TR^R6A; ΔN10aa: CifR_TR lacking residues 1–10.

Figure 5.

Comparison of DNA-bound CifR_TR and apo-CifR_TR structures. (A) Overall structure of apo-CifR_TR. The structure of apo-CifR_TR is shown as blue ribbons. The distance between the two Pro45 residues in the CifR dimer is shown. (B) Superposition of the CifR_TR:dsDNA_26L complex and the apoCifR_TR dimer. C_α/no outlier least-squares superposition of apo-CifR_TR (blue ribbons) and DNA-bound CifR_TR (green ribbons; orange DNA) performed in PyMol results in an RMSD of 3.1 Å. (C) Superposition of CifR_TR:dsDNA_26L complex and apo-CifR_TR aligned at the ligand-binding domain of one protomer. A 90° rotation of the view in (B) results in the view shown here. Only one protomer is shown for clarity. (D) Conformational change of helix α4. Alignment of apo-CifR_TR and DNA-bound CifR_TR using the DNA-binding domain of one protomer of each dimer reveals the conformational change of helix α4 upon DNA binding. Only helices α1-α4 are shown for clarity. (E) Conformational change of helix α6. Alignment of the apo-CifR_TR and DNA-bound CifR_TR using the ligand-binding domain of one protomer of each dimer reveals the conformational change of helix α6 upon DNA binding. Only helices α5-α7 are shown for clarity.

Figure 6.

The role of conserved residue Cys107 in the potential epoxide-binding pocket. (A) The potential epoxide-binding pocket in CifR_TR. Only one CifR_TR protomer is shown in the CifR_TR:dsDNA_26L structure for clearer view of the potential epoxide-binding pocket formed by helices α5 - α7 (green); other helices are shown in grey. (B) A closeup view of the proposed epoxide-binding pocket. Cys107 lies at the bottom of the epoxide-binding pocket (orange stick figure). (C) Response of CifR_TR Cys107 mutants to EBH. The involvement of Cys107 in ligand recognition and/or transduction of ligand binding to DNA release was assessed by EMSA with CifR_TR, CifR_TR^C107S, and CifR_TR^C107T mutants. The DNA concentration was 0.5 μM. Cpd = compound. Solvent (DMSO) alone did not disrupt protein-DNA complexes (not shown). (D) Response of CifR_TR protein to NEM analyzed by EMSA. A mixture containing 0.5 μM DNA and 1 μM CifR_TR was titrated with 0.5, 1, 2 and 4 mM EBH or the cysteine-modifying compound NEM. (E) Comparison of the ligand-binding pockets of CifR_TR:dsDNA_26L and CifR_TR^C107S:dsDNA_26L. DNA-bound CifR_TR (green sticks) and DNA-bound CifR_TR^C107S (light grey sticks) were aligned using the ligand-binding domains. (F) and (G) mF_O-DF_C maps of the ligand-binding pockets of DNA-bound CifR_TR (green sticks) and DNA-bound CifR_TR^C107S (grey sticks). Positive peaks are shown as green mesh for CifR_TR:dsDNA_26L and grey mesh for CifR_TR^C107S:dsDNA_26L. Both maps are contoured to 3.0σ.

Figure 7.

Recognition of operator DNAs by TFRs. (A) and (B) Proteins are shown as blue ribbons. DNA is shown in black and grey for each strand of the DNA duplex. The solid ovals indicate the two-fold rotational symmetry with the dotted lines indicating the orientation of the axes. The arrows below indicate the inverted repeats of DNA. (A) Structure of DNA-bound TetR. The center of the DNA is shown in red. (B) Structure of DNA-bound QacR. The rotation axis is perpendicular to the plane of view. The inset shows a 90°-rotated view of the main panel. (C) Operator DNA sequences of 21 TFRs with known DNA-complexed structure. The operator DNA sequences analyzed here are those used in the protein-DNA complex structure determination. The predicted center of each inverted repeat is shown with red text or bars. Structures in which only half of the operator DNA was used in the crystallization are labeled with an asterisk. The bases with potential inverted repeats property are underlined and in bold. The centers for two embedded repeats are highlighted in grey. The embedded repeats are shown in detail in Fig. S11. (D) Operator DNA sequences of CifR. The bases with potential inverted repeats property are underlined and in bold. The conserved residues between the two operator DNA sequence are marked by vertical alignment bars (black).