Crystal Structures of Multidrug Binding Protein TtgR in Complex with Antibiotics and Plant Antimicrobials

Abstract

Antibiotic resistance is a widely spread phenomenon. One major mechanism that underlies antibiotic resistance in bacteria is the active extrusion of toxic compounds through the membrane-bound efflux pumps that are often regulated at the transcriptional level. TtgR represses the transcription of TtgABC, a key efflux pump in Pseudomonas putida, which is highly resistant to antibiotics, solvents and toxic plant secondary products. Previously we showed that TtgR is the only reported repressor that binds to different classes of natural antimicrobial compounds, which are also extruded by the efflux pump. We report here five high-resolution crystal structures of TtgR from the solvent-tolerant strain DOT-T1E, including TtgR in complex with common antibiotics and plant secondary metabolites. We provide structural basis for the unique ligand binding properties of TtgR. We identify two distinct and overlapping ligand binding sites; the first one is broader and consists of mainly hydrophobic residues, whereas the second one is deeper and contains more polar residues including Arg176, a unique residue present in the DOT-T1E strain but not in other Pseudomonas strains. Phloretin, a plant antimicrobial, can bind to both binding sites with distinct binding affinities and stoichiometries. Results on ligand binding properties of native and mutant TtgR proteins using isothermal titration calorimetry confirm the binding affinities and stoichiometries, and suggest a potential positive cooperativity between the two binding sites. The importance of Arg176 in phloretin binding was further confirmed by the reduced ability of phloretin in releasing the mutant TtgR from bound DNA compared to the native protein. The results presented here highlight the importance and versatility of regulatory systems in bacterial antibiotic resistance and open up new avenues for novel antimicrobial development.

Crystal structure of the transcriptional regulator Rv1219c of Mycobacterium tuberculosis

“…This was clearly demonstrated by the structures of the QacR 18,26 and Rv3066 14 regulators. A similar characteristic for the TetR-family regulators that recognize negatively charged antimicrobials has also been observed with TtgR 27 and CmeR. 28 In this case, positively charged histidines or lysines within the ligand-binding pockets are critical for the binding.…”

The crystal structure of multidrug-resistance regulator RamR with multiple drugs

“…In this study, we identified five substrates of the RamR protein, including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6G. Similar approaches in crystallizing the TetR family regulators with multiple drugs have been also reported in QacR 20 , TtgR 21 ARTICLE six amino acid substitutions in RamR that are related to bacterial drug resistance, including T18P, R46N, R46P, Y59H, M84I and E160D 15,17,23 . As shown in Supplementary Fig.…”

“…It was originally reported that all the ligands bind to QacR with a 1:2 stoichiometry (one ligand per QacR dimer), 20 whereas either 1:2 or 1:1 stoichiometry has been observed for RamR. Similar observations were reported in TtgR 21 . All the drugs studied, except dequalinium, were found completely buried in the RamR-binding pocket.…”

Crystal structures of CmeR‐bile acid complexes from Campylobacter jejuni

“…It is intriguing that the multidrug binding protein TtgR seems to utilize a different mechanism to recognize negatively charged antibiotics and plant antimicrobials. 21 The hydrophobic environment is provided in the ligand binding pocket at the C-terminal regulatory domain. In addition, a positively charged histidine and a polar asparagine are also found to involve in the binding.…”