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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

Far UV CD experiments of TtgRWT and variants in the absence of effectors.(A) Far UV CD spectra at 30°C. (B) Thermal unfolding of TtgRWT and mutants monitored by CD ellipticity at 222 nm. Variants are represented in grey line (TtgRWT), black solid line (TtgRH114A), black dashed line (TtgRN110A), black dotted line (TtgRE78A) and black squares and line (TtgRS77A).
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pone.0138469.g002: Far UV CD experiments of TtgRWT and variants in the absence of effectors.(A) Far UV CD spectra at 30°C. (B) Thermal unfolding of TtgRWT and mutants monitored by CD ellipticity at 222 nm. Variants are represented in grey line (TtgRWT), black solid line (TtgRH114A), black dashed line (TtgRN110A), black dotted line (TtgRE78A) and black squares and line (TtgRS77A).

Mentions: To analyze the conservation of the secondary structure of the variants, we carried out far-UV CD experiments on the native protein and mutants at 30°C (Fig 2A). The resulting spectra are similar, indicating that the overall secondary structure is not altered by mutations. The CD spectra were deconvoluted using the CDNN program, http://bioinformatik.biochemtech.uni-halle.de/cdnn/ [24], in order to determine the protein percentage in α-helix, beta-turn and random coil. The data revealed a similar percentage of α-helix, beta-turn and random coil for most of the variants, in comparison with TtgRWT (Table 1).


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)

Far UV CD experiments of TtgRWT and variants in the absence of effectors.(A) Far UV CD spectra at 30°C. (B) Thermal unfolding of TtgRWT and mutants monitored by CD ellipticity at 222 nm. Variants are represented in grey line (TtgRWT), black solid line (TtgRH114A), black dashed line (TtgRN110A), black dotted line (TtgRE78A) and black squares and line (TtgRS77A).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138469.g002: Far UV CD experiments of TtgRWT and variants in the absence of effectors.(A) Far UV CD spectra at 30°C. (B) Thermal unfolding of TtgRWT and mutants monitored by CD ellipticity at 222 nm. Variants are represented in grey line (TtgRWT), black solid line (TtgRH114A), black dashed line (TtgRN110A), black dotted line (TtgRE78A) and black squares and line (TtgRS77A).
Mentions: To analyze the conservation of the secondary structure of the variants, we carried out far-UV CD experiments on the native protein and mutants at 30°C (Fig 2A). The resulting spectra are similar, indicating that the overall secondary structure is not altered by mutations. The CD spectra were deconvoluted using the CDNN program, http://bioinformatik.biochemtech.uni-halle.de/cdnn/ [24], in order to determine the protein percentage in α-helix, beta-turn and random coil. The data revealed a similar percentage of α-helix, beta-turn and random coil for most of the variants, in comparison with TtgRWT (Table 1).

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