Crystal structure of Pseudomonas aeruginosa transcriptional regulator PA2196 bound to its operator DNA

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Highlights

  • The structure of PA2196: dsDNA complex revealed that two dimers bind to one dsDNA.

  • This binding mode is similar to that of QacR in that two dimers bind to one dsDNA.

  • Residues in the α3 helix mainly interact with the base and phosphate of dsDNA.

  • PA2196 undergoes large conformational changes upon DNA binding.

  • The structure of PA2196: dsDNA complex help understand the recognition mechanism.

Abstract

Pseudomonas aeruginosa is a major opportunistic human pathogen. PA2196 from P. aeruginosa is a member of TetR family of transcriptional repressors, which is involved in adaptation to environmental changes as well as bacterial antibiotic resistance. PA2196 consists of nine α-helical bundles divided into two separate domains. The N-terminal domain, called the DNA-binding domain, is composed of helices α1–α3 and has a helix-turn-helix motif. The C-terminal domain, called the ligand-binding domain, has a hydrophobic pocket for ligand binding. Here, PA2196 was shown to bind to a 25 bp semi-palindromic dsDNA located in the upstream region of its own gene. The crystal structure of the PA2196–25mer dsDNA complex determined at a resolution of 2.9 Å revealed that two dimers of PA2196 bound to one dsDNA, with each monomer interacting with the major groove of DNA. Especially, residues in helix α3, including Lys41, Gly42, Ser43, and Tyr45, interacted mainly with the base and phosphate backbone of dsDNA. PA2196 underwent large conformational changes upon DNA binding, as the distances between DNA-binding domains measured between two G42s in subunits A and B decreased from 41.7 Å to 36.8 Å. Our crystal structure of PA2196–25mer dsDNA complex revealed that PA2196 is similar to QacR in that two dimers bound to one dsDNA through specific interactions.

Introduction

Bacteria respond rapidly to variations in the environment by using regulatory proteins capable of detecting the changes [1]. Pseudomonas aeruginosa is a major opportunistic human pathogen, and its resistance to antibiotics and disinfectants are responsible for many diseases [2]. P. aeruginosa possesses TetR family repressors, which respond to specific environmental changes and induce target gene expression by being released from operator DNA upon ligand binding [1]. TetR family proteins share a common structural feature in the form of 10 α-helical bundles divided into two domains. The N-terminal domain, called the DNA-binding domain, is composed of helices α1–α3 and binds to palindromic operator DNA. Helix α3 plays an important role by binding specifically to the major groove of DNA. The C-terminal domain, called the ligand-binding domain, is composed of helices α4–α10 and contains a ligand-binding pocket of diverse size [3], [4]. For example, the C-terminal domain of TetR repressor proteins recognizes tetracyclin and induces conformational changes in the N-terminal domain leading to the release of the repressor [5]. After the TetR repressor is released from operator DNA, downstream genes are activated to remove tetracyclin by an efflux pump [6].

We have previously determined the structure of apo PA2196 that contains a HTH-motif in the DNA-binding domain and a hydrophobic pocket in the ligand-binding domain [4]. To examine the exact binding mechanism of PA2196 to DNA, we determined the structure of PA2196 bound to 25 bp semi-palindromic dsDNA region located upstream of the PA2196 gene. The structure revealed that two dimers of PA2196 bound to one semi-palindromic DNA, which is similar to the binding mode of QacR repressor [7]. Our structure also elucidated critical residues in DNA binding and conformational changes upon DNA binding.

Section snippets

Cloning and protein expression

The PA2196 gene was amplified from P. aeruginosa genomic DNA by polymerase chain reaction (PCR). The purified PCR product was cloned into pET28b vector using NheI and EcoRI restriction enzymes with an N-terminal His6-tag and thrombin-cut site. The construct was transformed into BL21 (DE3) Escherichia coli strain (Novagen). Cells were grown in LB media containing 30 μg/ml of kanamycin at 37 °C until an OD600 of 0.8. Protein expression was induced at 18 °C with 1 mM isopropyl β-d-1-thiogalacto

Overall structure of PA2196 with 25mer dsDNA

The crystal structure of PA2196 with 25mer dsDNA was determined at a 2.9 Å resolution by molecular replacement. The crystal structure revealed a protein–DNA complex composed of two pairs of dimers (cyan and green; magenta and yellow) bound to one 25mer dsDNA each (Fig. 1A and B). There were four such complexes in the asymmetric unit (total of eight dimers and four 25mer dsDNAs). The two dimers bound to the opposite side of the DNA with an angle of about 135° between them (Fig. 1B). The overall

Acknowledgments

We thank the staff members of PAL beamline 5C for assistance in data collection. This work is supported by Basic Science Research Program through the National Research Foundation of Korea (Grant Number: NRF-2013R1A1A2006096) and by the 2012 sabbatical year research grant of the University of Seoul.

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