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, 42 (12), 850-857

Crystal Structure of the Regulatory Domain of MexT, a Transcriptional Activator of the MexEFOprN Efflux Pump in Pseudomonas aeruginosa

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Crystal Structure of the Regulatory Domain of MexT, a Transcriptional Activator of the MexEFOprN Efflux Pump in Pseudomonas aeruginosa

Suhyeon Kim et al. Mol Cells.

Abstract

The Gram-negative opportunistic pathogen, Pseudomonas aeruginosa , has multiple multidrug efflux pumps. MexT, a LysR-type transcriptional regulator, functions as a transcriptional activator of the MexEF-OprN efflux system. MexT consists of an N-terminal DNA-binding domain and a C-terminal regulatory domain (RD). Little is known regarding MexT ligands and its mechanism of activation. We elucidated the crystal structure of the MexT RD at 2.0 Å resolution. The structure comprised two protomer chains in a dimeric arrangement. MexT possessed an arginine-rich region and a hydrophobic patch lined by a variable loop, both of which are putative ligand-binding sites. The three-dimensional structure of MexT provided clues to the interacting ligand structure. A DNase I footprinting assay of full-length MexT identified two MexT-binding sequence in the mexEF oprN promoter. Our findings enhance the understanding of the regulation of MexT-dependent activation of efflux pumps.

Keywords: MexT; Pseudomonas aeruginosa; antibiotic resistance; crystal structure; lysR-type transcriptional regulator.

Conflict of interest statement

Disclosure

The authors have no potential conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1. Molecular size of the purified proteins
(A) FL MexT. (B) MexT RD. Primary y-axis, molar mass determined using multi-angle light scattering (MALS; black line); secondary y-axis, concentration. Protein concentration was assessed by measuring the absorbance at 280 nm (red), light scattering (LS; green), and the refractive index (RI; purple). X-axis, elution time from size exclusion chromatography.
Fig. 2
Fig. 2. Structure of MexT RD dimer
Chains A–D of the MexT RD dimers are colored purple, green, cyan, and yellow, respectively, with secondary structures labeled accordingly. RD-I comprises three α-helices (α1, α2, and α7), and five β-strands (β1, β2, β3, β4, and β10). RD-II comprises four α-helices (α3, α4, α5, and α6), and five β-strands (β5, β6, β7, β8, and β9). Red line, RD-I; blue line, RD-II.
Fig. 3
Fig. 3. Dimeric arrangement of MexT RD
(A) Sulfates in the A:B chain dimer. A sulfate ion (Sul1) is bound to Arg220 and Arg269 of chain B. The other sulfate ion (Sul2) interacts with Arg134 of chain A (purple) and Arg135 of chain B (green; bottom panel). (B) Sulfates in the C:D chain dimer. Two sulfate ions (Sul3 and Sul4) are bound. Red squares, sulfates bound to arginine residues; the main residues involved are marked. Purple ovals, two fold enlargements of detail. (C) Oxidized (active) OxyR RD from E. coli (PDB code, 1I6A; blue) superimposed on the MexT RD (green). (D) Reduced (inactive) OxyR RD from E. coli (PDB code, 1A69; orange) superimposed on the MexT RD (green).
Fig. 4
Fig. 4. Putative ligand binding sites and variable (V)-loops
(A) Conformation of the V-loop in each monomer. Red lines represent V-loops; a red dotted line represents a disordered region of chain D. A stick model was generated to show positively charged and hydrophobic residues with chain B. (B) The pocket at the putative ligand-binding site. The pocket at the putative ligand binding site in chain B is shown with a sulfate ion on the surface. A stick model was generated to show positively charged and hydrophobic residues. The hydrophobic patch is lined.
Fig. 5
Fig. 5. MexT binding sequences identified by DNase I footprinting
(A) A 630-bp DNA fragment containing the mexT-mexE intergenic region was labeled with 6-fluorescein amidite and then used as a DNA probe. The fluorescence-labeled DNA probe (400 ng) was incubated without or with 200 ng of purified FL MexT. The regions protected by MexT are indicated by red squares (MexT binding site I and II). Nucleotide numbers shown are the relative positions when the transcription start site mexE is designated as +1. (B) Comparison of the MexT-binding sequences. The two putative MexT-binding sequences in the mexEF-oprN regulatory region are shown at the top. The MexT-binding sequences from other promoters of MexT regulons are aligned below. Nucleotides in bold and underlined represent a partially conserved palindromic sequence, suggesting the potential consensus MexT-binding sequence, ATCA(N)7CGAT.

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