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Molecular Binding Mechanism of TtgR Repressor to Antibiotics and Antimicrobials.

Fernandez-Escamilla AM, Fernandez-Ballester G, Morel B, Casares-Atienza S, Ramos JL - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Environmental Protection Department, Estación Experimental del Zaidín (EEZ), Spanish National Research Council (CSIC), C/ Profesor Albareda, 1, E-18008 Granada, Spain.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Effect of naringenin and phloretin on the dissociation of TtgRWT and variants (TtgRH114A, TtgRN110A and TtgRE78A) from ttgR-ttgABC intergenic region by EMSA.(-) 1nM of free labeled operator DNA, (+) DNA-complex with 2 μM of TtgRWT and its variants, and DNA complex in the presence of 5μM of naringenin (Nar) and phloretin (Phlr).
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pone.0138469.g005: Effect of naringenin and phloretin on the dissociation of TtgRWT and variants (TtgRH114A, TtgRN110A and TtgRE78A) from ttgR-ttgABC intergenic region by EMSA.(-) 1nM of free labeled operator DNA, (+) DNA-complex with 2 μM of TtgRWT and its variants, and DNA complex in the presence of 5μM of naringenin (Nar) and phloretin (Phlr).

Mentions: As expected, the binding of the effector to the TtgR repressor induced the dissociation of the repressor-operator complex. It has previously been described that phloretin and naringenin cause the dissociation of the repressor-operator complex [13]. To evaluate the effects of the different ligands on the effector-mediated DNA operator dissociation, we performed electrophoretic mobility shift assays (EMSA) (Fig 5). Our results revealed that, in absence of effectors, most of the variants (except TtgRS77A) were able to bind to and retard the DNA fragment, containing the ttgABC-ttgR intergenic region. In the presence of phloretin, TtgRE78A and TtgRH114A released the DNA completely while TtgRN110A only dissociated 66% of DNA. In the case of naringenin, TtgRE78A and TtgRH114A released around 30% of DNA while TtgRN110A was completely dissociated from the complex. These results suggest that mutations affect the binding or the accessibility of the effectors to the binding pocket and subsequently produce an effect on the repressor-operator complex dissociation.


Molecular Binding Mechanism of TtgR Repressor to Antibiotics and Antimicrobials.

Fernandez-Escamilla AM, Fernandez-Ballester G, Morel B, Casares-Atienza S, Ramos JL - PLoS ONE (2015)

Effect of naringenin and phloretin on the dissociation of TtgRWT and variants (TtgRH114A, TtgRN110A and TtgRE78A) from ttgR-ttgABC intergenic region by EMSA.(-) 1nM of free labeled operator DNA, (+) DNA-complex with 2 μM of TtgRWT and its variants, and DNA complex in the presence of 5μM of naringenin (Nar) and phloretin (Phlr).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4589371&req=5

pone.0138469.g005: Effect of naringenin and phloretin on the dissociation of TtgRWT and variants (TtgRH114A, TtgRN110A and TtgRE78A) from ttgR-ttgABC intergenic region by EMSA.(-) 1nM of free labeled operator DNA, (+) DNA-complex with 2 μM of TtgRWT and its variants, and DNA complex in the presence of 5μM of naringenin (Nar) and phloretin (Phlr).
Mentions: As expected, the binding of the effector to the TtgR repressor induced the dissociation of the repressor-operator complex. It has previously been described that phloretin and naringenin cause the dissociation of the repressor-operator complex [13]. To evaluate the effects of the different ligands on the effector-mediated DNA operator dissociation, we performed electrophoretic mobility shift assays (EMSA) (Fig 5). Our results revealed that, in absence of effectors, most of the variants (except TtgRS77A) were able to bind to and retard the DNA fragment, containing the ttgABC-ttgR intergenic region. In the presence of phloretin, TtgRE78A and TtgRH114A released the DNA completely while TtgRN110A only dissociated 66% of DNA. In the case of naringenin, TtgRE78A and TtgRH114A released around 30% of DNA while TtgRN110A was completely dissociated from the complex. These results suggest that mutations affect the binding or the accessibility of the effectors to the binding pocket and subsequently produce an effect on the repressor-operator complex dissociation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Environmental Protection Department, Estación Experimental del Zaidín (EEZ), Spanish National Research Council (CSIC), C/ Profesor Albareda, 1, E-18008 Granada, Spain.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus