Crystal structure of QscR, a Pseudomonas aeruginosa quorum sensing signal receptor

Abstract

Acyl-homoserine lactone (AHL) quorum sensing controls gene expression in hundreds of Proteobacteria including a number of plant and animal pathogens. Generally, the AHL receptors are members of a family of related transcription factors, and although they have been targets for development of antivirulence therapeutics there is very little structural information about this class of bacterial receptors. We have determined the structure of the transcription factor, QscR, bound to N-3-oxo-dodecanoyl-homoserine lactone from the opportunistic human pathogen Pseudomonas aeruginosa at a resolution of 2.55 Å. The ligand-bound QscR is a dimer with a unique symmetric "cross-subunit" arrangement containing multiple dimerization interfaces involving both domains of each subunit. The QscR dimer appears poised to bind DNA. Predictions about signal binding and dimerization contacts were supported by studies of mutant QscR proteins in vivo. The acyl chain of the AHL is in close proximity to the dimerization interfaces. Our data are consistent with an allosteric mechanism of signal transmission in the regulation of DNA binding and thus virulence gene expression.

Fig. 1.

Structure of QscR bound to 3OC12-HSL. (A) Ribbon representation shows chain A (cyan), chain B (green), 3OC12-HSL (orange), with oxygen atoms in red and the nitrogen atom in blue. The LBD, the DBD, the first visible amino acid, and the C terminus are indicted for each chain. (B) Electron density map surrounding 3OC12-HSL in chain A. The σ_a-weighted 2Fo-Fc map contoured at 1.5σ is shown in gray, with the underlying model of QscR (cyan).

Fig. 2.

QscR dimerization interactions. (A) Subset of the predicted intermolecular contacts between the LBDs of chain A (cyan) and B (green). Ionic interactions are shown between both Glu84 with Lys121, and Tyr85 forms van der Waal’s contacts with the opposite LBD. This interface is near the acyl-chain region of the AHL-binding pocket (AHL is in tan). (B) Intermolecular interactions at the interface of LBD-A with DBD-B and LBD-B. Each chain is colored as in A. Hydrogen-bonding contacts are shown between Arg42 and Arg79 with the carboxylate group of Asn237. This interface lies near the acyl-chain region of the AHL-binding pocket. (C) Activity assays in E. coli containing the QscR expression vector pJN105Q and the PA1897-lacZ vector pJL101 comparing the wild-type QscR (WT) to four of the substitutions of key residues noted in A and B. Each assay was conducted at three different concentrations of AHL (10, 50, and 250 nM for 3OC12-, C12-, and 3OC14-HSLs and 10, 50, and 250 μM for 3OC6-HSL). Activity of WT QscR in the presence of 250 nM 3OC12-HSL was set as 100%. Values are means of three independent experiments and bars show standard deviations.

Fig. 3.

AHL-binding pocket interactions. (A) Hydrogen-bonding contacts between 3OC12-HSL and QscR. Water molecules are shown as red spheres. Dashed lines indicate presumed hydrogen bonds. (B) Hydrophobic interactions formed between the aliphatic tail of 3OC12-HSL and QscR. (C and D) Activity assays comparing the wild-type QscR (WT) to five of the substitutions of key residues noted in A and B. Each assay was conducted at the same three different concentrations of AHL shown in Fig. 2. Values are means of three independent experiments, and bars show standard deviations.

Fig. 4.

Ligand binding and dimerization of QscR compared to other AHL receptors. (A) Ligands in the superimposed QscR (cyan), LasR (purple), TraR (gold), SdiA (blue), and CviR (green). (B) The LBD dimer of QscR (cyan) superimposed on LasR (purple), TraR (gold), and CviR (green). AHLs are not shown. The orientation of the LBDs in QscR and LasR are similar, but for TraR one of the LBDs is rotated nearly 150° relative to QscR, and for CviR, one LBD is rotated 43° in the opposite direction and is shifted by nearly 30 Å.

Fig. 5.

Model of DNA recognition by QscR. (A) Superposition of the QscR-AHL complex (cyan) onto the DBD and DNA from the TraR–AHL–DNA complex (in gold). (B) CD spectra of QscR with 3OC12-HSL and QscR with 3OC12-HSL and DNA. (C) Thermal melting monitored by CD of QscR in the presence of 3OC12-HSL, with and without DNA.