Structural and biochemical analysis of the pentapeptide repeat protein EfsQnr, a potent DNA gyrase inhibitor - PubMed (original) (raw)

Structural and biochemical analysis of the pentapeptide repeat protein EfsQnr, a potent DNA gyrase inhibitor

Subray S Hegde et al. Antimicrob Agents Chemother. 2011 Jan.

Abstract

The chromosomally encoded Qnr homolog protein from Enterococcus faecalis (EfsQnr), when expressed, confers to its host a decreased susceptibility to quinolones and consists mainly of tandem repeats, which is consistent with belonging to the pentapeptide repeat family of proteins (PRPs). EfsQnr was cloned with an N-terminal 6× His tag and purified to homogeneity. EfsQnr partially protected DNA gyrase from fluoroquinolone inhibition at concentrations as low as 20 nM. EfsQnr inhibited the ATP-dependent supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC(50)) of 1.2 μM, while no significant inhibition of ATP-independent relaxation activity was observed. EfsQnr was cytotoxic when overexpressed in Escherichia coli, resulting in the clumping of cells and a loss of viability. The X-ray crystal structure of EfsQnr was determined to 1.6-Å resolution. EfsQnr exhibits the right-handed quadrilateral beta-helical fold typical of PRPs, with features more analogous to MfpA (mycobacterium fluoroquinolone resistance pentapeptide) than to the PRPs commonly found in cyanobacteria.

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Figures

FIG. 1.

FIG. 1.

Inhibition of DNA gyrase by _Efs_Qnr. Inhibition of supercoiling (A) and relaxation activities (B) of DNA gyrase by _Efs_Qnr. (A) Lane 1, relaxed plasmid pBR322 alone; lane 2, relaxed pBR322 plus 3 U of gyrase; lanes 3 to 8, 3 U of gyrase and 0.25, 0.5, 1, 2, 5, and 10 μM _Efs_Qnr, respectively. (B) Lane 1, supercoiled plasmid pBR322 alone; lane 2, supercoiled pBR322 plus 50 μM _Efs_Qnr; lane 3, supercoiled pBR322 plus 25 U of gyrase; lanes 4 to 8, 25 U of gyrase and 5, 10, 25, 50, and 100 μM _Efs_Qnr, respectively. nc, l, and sc represent nicked circular, linear, and supercoiled forms, respectively.

FIG. 2.

FIG. 2.

Inhibition of supercoiling activity by ciprofloxacin and partial protection by _Efs_Qnr. (A) Lane 1, relaxed plasmid pBR322 alone; lane 2, relaxed pBR322 plus 3 U of gyrase; lanes 3 to 8, 3 U of gyrase and 0.1, 0.25, 0.5, 1, 2, and 5 10 μM ciprofloxacin in the absence _Efs_Qnr (top) and in the presence of 0.2 μM _Efs_Qnr (bottom), respectively. (B) Graphical representation of the inhibition data. Symbols are experimentally determined values, while the smooth lines are the fit of the data.

FIG. 3.

FIG. 3.

Scanning electron microscopy of E. coli BL21(DE3) cells expressing _Efs_Qnr. (A) Control cells; (B) 2 h after induction of _Efs_Qnr expression; (C) 8 h after induction. (D) Effect of _Efs_Qnr induction on the viability of E. coli. Cultures were diluted identically and plated onto LB plates containing 30 μg/ml kanamycin. Bottom, control; top, cells expressing _Efs_Qnr.

FIG. 4.

FIG. 4.

Primary sequence of _Efs_Qnr and _Mt_MfpA. Pentapeptide repeats are compiled into four columns (gray-boxed residues) indicating their location within the four faces of the quadrilateral β-helix. Pentapeptide residue types (i.e., i+1, i+2, etc.) are shown between the two sequences. Helices are displayed in salmon-colored boxes. Residues that support the largest deviations from the standard β-helix are shown in blue.

FIG. 5.

FIG. 5.

Monomer structure of _Efs_Qnr and _Mt_MfpA. (A) Ribbon diagram of _Efs_Qnr and _Mt_MfpA. β-Helical structural elements are colored by face type as aligned in Fig. 4. α-Helices are in salmon, and the N-terminal extension of _Efs_Qnr is black. Coils have repeats that are colored in the order green, cyan, yellow, and red. Residues within repeats that exhibit full intercoil hydrogen bonding are shown as strands and are indicative of the following turn being type IV, while β-bridges are illustrated as spheres and are indicative of the following turn being type II. (B) Stereoview of the superposition of _Efs_Qnr (black trace) and _Mt_MfpA (orange trace).

FIG. 6.

FIG. 6.

Molecular dimer of _Efs_Qnr. Ribbon diagram of the _Efs_Qnr and _Mt_MfpA dimer orientated based on the superposition of _Mt_MfpA on subunit B of _Efs_Qnr.

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