Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands

Abstract

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors.

Figure 1.

Determination of the ‘minimal ligand binding site’ of EthR. Crystal structure of the ligand binding site of six holoforms of EthR: (a) EthR-hexadecyl-octanoate (PDB#1U9N), (b) EthR-Dioxane (PDB#1T56), (c) EthR-BDM31381 (PDB#3G1M), (d) EthR-BDM14801 (PDB#3O8G), (e) EthR-BDM31343 (3TP0) and (f) EthR-BDM33066 (PDB#3Q0W). Close-views of the occupancy of each ligand in the binding pocket of EthR. The protein is displayed in ribbon and the side chain of Asn179 is represented by sticks colored according the following atom color schemes: carbon in blue, oxygen in red and nitrogen in dark blue. When observed, hydrogen bonds with this latter side chain are depicted by dashed lines with the interacting distance indicated. (g) Superimposition of the six crystal structures. The cyclic moieties of BDM14801 (orange), BDM31381 (red), BDM33066 (dark blue), BDM31343 (light blue), the C₁₈–C₂₀ portion of hexadecyl-octanoate (yellow) and the upper dioxane (pink) share a narrow space delimited with the gray translucent disk.

Figure 2.

In silico mutagenesis model of G106W. (a) Front and 90° lateral views of the superimposition of the ligands in their respective position in the ligand binding pocket of EthR. The gray rectangle highlights the zone common to all ligands. (b) Front and 90° lateral views of the in silico substitution of glycine 106 for tryptophan.

Figure 3.

Crystallographic structure of EthR_G106W. (a) Close view of the indole group of tryptophan 106 as observed in the crystal structure of the variant EthR_G106W (PDB#3TP3). (b) Close view of the same region superimposed with the crystal structure of EthR-BDM 33066 (ligand BDM 33066 is shown in purple). (c) Superimposition of EthR_G106W (Gold) and EthR-hexadecyl-octanoate (Pink). Only selected amino acid side chains are shown. Reorganization of amino acid side chains in the north part (upper panel) and in the south part (lower panel) of the ligand binding pocket. (d) Measurements of the distance separating the two helix-turn-helix DNA-binding motifs (α carbon of P59's) in the variant EthR_G106W.

Figure 4.

Binding of EthR and EthR_G106W to the DNA operator of ethA measured by surface plasmon resonance. Sensorgram showing two successive injections of 0.3 µM EthR (at 500 and 1000 s) on a sensor chip functionalized with 250 arbitrary RU of the biotinylated intergenic ethA-R DNA region (9), followed by the injection of 0.3 µM EthR_G106W (at 1600 s). A third injection of 0.3 µM EthR was done (at 2100 s) to control the binding capacity of the chip after the previous injection of EthR_G106W. Arrows indicate time of protein injection.

Figure 5.

Binding of ligands to EthR and EthR_G106W. (a) Thermal stability of EthR (dashed curve) and EthR_G106W (plain curve). Curves correspond to the first derivative of the fluorescence of Sypro Orange recorded during samples denaturation (30 values per °C). Melting temperature (T_m) for each sample corresponds to the maxima of the curve. (b) T_m of EthR and EthR_G106W in the presence of indicated ligands. (c) Isothermal Titration Calorimetry study of the binding of BDM14801 to EthR (left) and EthR_G106W (right). The graph shows raw data of a typical experiment, expressed in µcal/s versus time units.

Figure 6.

Upfield region of the 1D proton spectrum of apo-EthR (bottom), EthR + ligand BDM31381 (middle) and apo-EthR_G106W (top). Signals in this region correspond to methyl protons whose resonance is shifted out of the crowded methyl region (1.5–0.8 ppm) by the ring current effect of nearby aromatics (Full NMR spectra are shown in Supplementary Figure S2). Prediction by ShiftX2 (35) shows plausible candidates (Supplementary Figure S1). Whereas these resonances are severely broadened in the apo-EthR spectrum, they sharpen significantly upon ligand addition or in the G106W mutant. Resonances with conserved frequencies have been indicated by dotted vertical lines. Differences between the most shifted resonances in the EthR-ligand and EthR_G106W mutant could come from a differential magnetic influence of the ligand and the Trp side chain, and cannot necessarily be interpreted in terms of structural differences.