Molecular Binding Mechanism of TtgR Repressor to Antibiotics and Antimicrobials

A disturbing phenomenon in contemporary medicine is the prevalence of multidrug-resistant pathogenic bacteria. Efflux pumps contribute strongly to this antimicrobial drug resistance, which leads to the subsequent failure of clinical treatments. The TtgR protein of Pseudomonas putida is a HTH-type transcriptional repressor that controls expression of the TtgABC efflux pump, which is the main contributor to resistance against several antimicrobials and toxic compounds in this microbe. One of the main strategies to modulate the bacterial resistance is the rational modification of the ligand binding target site. We report the design and characterization of four mutants-TtgRS77A, TtgRE78A, TtgRN110A and TtgRH114A - at the active ligand binding site. The biophysical characterization of the mutants, in the presence and in the absence of different antimicrobials, revealed that TtgRN110A is the variant with highest thermal stability, under any of the experimental conditions tested. EMSA experiments also showed a different dissociation pattern from the operator for TtgRN110A, in the presence of several antimicrobials, making it a key residue in the TtgR protein repression mechanism of the TtgABC efflux pump. We found that TtgRE78A stability is the most affected upon effector binding. We also probe that one mutation at the C-terminal half of helix-α4, TtgRS77A, provokes a severe protein structure distortion, demonstrating the important role of this residue in the overall protein structure and on the ligand binding site. The data provide new information and deepen the understanding of the TtgR-effector binding mechanism and consequently the TtgABC efflux pump regulation mechanism in Pseudomonas putida.This work was supported by Spanish Ministry of Economy and Competitiveness, National programme for Recruitment and Incorporation of Human Resources, Subprogramme: Ramon y Cajal RYC-2009-04570 and grant P11-CVI-7391 from Junta de Andalucía and EFDR (European Regional Development Fund)

Effect of naringenin and phloretin on the dissociation of TtgR^WT and variants (TtgR^H114A, TtgR^N110A and TtgR^E78A) from <i>ttgR-ttgABC</i> intergenic region by EMSA.

<p>(-) 1nM of free labeled operator DNA, (+) DNA-complex with 2 μM of TtgR^WT and its variants, and DNA complex in the presence of 5μM of naringenin (Nar) and phloretin (Phlr).</p

Thermodynamic parameters of the association of TtgR and mutants with chloramphenicol, naringenin and phloretin.

<p>Thermodynamic parameters of the association of TtgR and mutants with chloramphenicol, naringenin and phloretin.</p

Far UV CD experiments of TtgR^WT and variants in the absence of effectors.

<p>(A) Far UV CD spectra at 30°C. (B) Thermal unfolding of TtgR^WT and mutants monitored by CD ellipticity at 222 nm. Variants are represented in grey line (TtgR^WT), black solid line (TtgR^H114A), black dashed line (TtgR^N110A), black dotted line (TtgR^E78A) and black squares and line (TtgR^S77A).</p

Structural contents of TtgR ^WT and mutants (%), in the absence of effectors.

<p>Data were obtained by deconvolution of the far UV CD spectra at 30°C.</p

Sequence alignment of close homologues of TtgR (<i>Pseudomonas putida</i> DOT-T1E).

<p>Mutated residues (S77, E78, N110 and H114) are boxed and shaded.</p

Thermal denaturation followed by DSC of TtgR^WT and variants (TtgR^H114A, TtgR^N110A, TtgR^E78A and TtgR^S77A) in the presence and in the absence of ligands (black solid lines free; grey solid lines 250 μM of chloramphenicol; grey dashed lines 250 μM naringenin; grey dotted lines 250 μM phloretin).

<p>Thermal denaturation followed by DSC of TtgR^WT and variants (TtgR^H114A, TtgR^N110A, TtgR^E78A and TtgR^S77A) in the presence and in the absence of ligands (black solid lines free; grey solid lines 250 μM of chloramphenicol; grey dashed lines 250 μM naringenin; grey dotted lines 250 μM phloretin).</p

ITC isotherms for the binding of TtgR^E78A mutant to: A) chloramphenicol, B) naringenin and C) phloretin.

<p>Experiments were performed as described in “Materials and Methods section” at 30°C with injections of the effector diluted in dialysis buffer into the protein. Upper panels: Heats changes for injections. Lower panels: Experimental heats measured for each effector injection. The curves correspond to the best fit using “one type of sites” model.</p

Thermodynamic parameters obtained by DSC and CD.

<p>Thermodynamic parameters for the thermal unfolding of TtgR^WT and variants (TtgR^H114A, TtgR^N110A, TtgR^E78A and TtgR^S77A) obtained from DSC and far-UV CD measurements. Internal calibrations of the CD and DSC equipments give errors in Tm of ± 0.20°C and ± 0.10°C respectively.</p

Theoretical energy measurements of models.

<p>*The values are presented in ΔΔG (kcal/mol) as the difference between the free energy of mutants with respect to the free energy of wild type. <b>Effector free</b>: stability energy calculated as the difference between folded and unfolded states. <b>With effectors</b>: free energy of binding calculated as the difference between the bound and unbound state.</p><p>Theoretical energy measurements of models.</p