EthR is a transcriptional regulator of the TetR family. EthR regulates the production of EthA, a monooxygenase implicated in the bioactivation of the antimycobacterial prodrug ETH. Our previous works showed that synthetic compounds mimicking fortuitous ligands of EthR inhibit EthR, and consequently, increase ETH bioactivation through the overproduction of EthA. Inhibition of the DNA binding function of EthR by these ligands follows a mode of action typical in the TetR family of repressors. In these dimeric regulators, invasion of the binding pockets by specific ligands results in the structural modifications of the helix-turn-helix motifs of each monomer, which translates in the loss of DNA recognition and binding to the repressor. The allosteric effect of the ligand, meaning the structural mechanism by which binding of molecules in the ligand pocket lead to the structural organization of distant motifs, is still not fully understood.
In the case of EthR, highly diverse ligands provoke the structural modifications leading to a conformation incompatible with DNA binding. This was shown with molecules as different as the 24-carbon long hexadecyl octanoate, small cyclic molecules such as dioxane, or various structure-based designed synthetic inhibitors. Here, we aligned liganded-EthR structures in order to identify structural similarities that may explain how chemically diverse ligands exert a common inhibitory effect on EthR. Interestingly, the common portion of the binding pocket of EthR that is in contact with every ligand is very limited. In addition, intensive structure activity relationship studies done to improve our synthetic ligands demonstrated that important structural constrains are required to allow ligand compatibility in this region (14–16, M. Flipo et al., submitted for publication). The goal of the actual study was to test whether this limited zone of the pocket could be the hot-spot leading to the allosteric response observed upon ligand binding, which impact the spatial architecture of the helix-turn-helix motifs of the repressor. Mutagenesis of glycine 106 to tryptophan was introduced in order to mimic the effect of ligands in this region of the pocket. This mutation entailed structural modifications and thermostabilization of the repressor equivalent to what was observed in the presence of ligands.
First, X-ray structural analysis revealed that the HTH DNA binding motifs of the mutated repressor are in a conformation incompatible with DNA binding. Indeed, EthRG106W
shows a structural spacing of its HTH domains (42.3
Å) in the range observed in ligand–protein complexes (42
Å with BDM31343 to 47
Å with BDM14801) reinforcing the idea of mechanistic mimicry between mutation G106W and ligands. Consistently, the mutated repressor showed a complete incapacity to bind to its DNA operator. Nevertheless, in their apo-form that is by nature competent for DNA binding, the majority of the regulators of the TetR family crystallizes in a conformation clearly not compatible with DNA binding (37
). These observations led some authors to postulate that crystal structures of apo-repressors of the TetR family reflect only a snap shot of the conformational repertoire of the proteins in solution and that the role of ligands could be to stabilize one conformation of the repertoire in which the recognition helices are too far apart to simultaneously bind to adjacent major groove of DNA (31
If true, this stabilization should naturally translate in some rigidification of the protein upon binding of ligand. Interestingly, the EthRG106W
crystal reveals the first apo form of EthR. Our inability to succeed in the crystallization of apo-form of the wild-type EthR suggest that the major effect of the mutation, mimicking ligands, was to improve the global stability of the protein. Preliminary NMR experiments are in support of this hypothesis, showing that a number of methyls of the protein have indeed their NMR signal affected by exchange broadening, reflecting typical dynamic processes on the micro- to millisecond time scale, but both the presence of ligand and the G106W mutation quench this dynamics (; Supplementary Figures S1
). From a thermodynamic point of view, fast internal dynamics of the ligand binding pocket of EthR have the potential to report on the number of states that the protein explores, hence, acting as an indirect measure of the residual entropy of the folded state. This observation could somehow remind the transcription factor ETS, for which flexibility was shown to be directly correlated to its capacity of binding to its DNA target (36
Alternatively, the observed distance of >42
Å that separate the HTH motifs in either EthRG106W
or in EthR–ligand complexes may by itself explain the inability of the repressor to bound to its DNA operator, and the drastic diminution of the internal flexibility observed in both cases could induce a locking mechanism to restrict further conformational change, as proposed recently by Le et al.
) for SimR.
Intensive mutagenesis studies on TetR have led to the identification of many variants that are no longer able to bind to DNA, whereas there still show good affinity for tetracycline (39
). Conversely, other variants that bind DNA more strongly in the presence of tetracycline have been described (41–44
). Here, we have identified a variant of EthR harboring the yet undescribed property to mimic the allosteric effect of known ligands, resulting in the complete inhibition of its DNA binding capacity. This mutation not only mimics the activity of small molecule ligands either at the functional level (inhibition of DNA binding propensity), but also at the structural level (equivalent structural conformations), or at the dynamic level (rigidification of the structure).
The design and improvement of high-affinity inhibitors in the context of drug discovery is a complex matter as the increase of enthalpy through increased bond strength is countervailed by the entropy reduction due to the global increase of order of the system. In allosteric systems, it is then essential to localize precisely the region of a protein implicated in ligand-induced signal in order to facilitate the design of structure-based inhibitors and maximize their entropic and enthalpic contributions. The present work used the large chemical and structural diversity of known EthR's ligands to identify one region of the protein implicated in the allosteric response of its DNA binding domain. Here, we have demonstrated that the inhibitory effect of highly diverse specific ligands of EthR can be mimicked by the mutation G106W. This now explains how two small cyclic molecules such as dioxane (12
) induced the same conformational state as the one induced with hexadecyl-octanoate (3
). Conversely, our results suggest that exploitation of deeper portions of the ligand pocket to increase their affinity/specificity would be successful as long as these molecules interact with the upper region of the pocket surrounding glycine 106.