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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

Thermal denaturation followed by DSC of TtgRWT and variants (TtgRH114A, TtgRN110A, TtgRE78A and TtgRS77A) 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).
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pone.0138469.g004: Thermal denaturation followed by DSC of TtgRWT and variants (TtgRH114A, TtgRN110A, TtgRE78A and TtgRS77A) 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).

Mentions: To characterize the binding of the effectors to TtgRWT and its variants as well as to describe the extent of the effect of each ligand on the structure and stability of the proteins, we performed temperature induced unfolding experiments on all of the variants using DSC and CD in the presence of naringenin, phloretin and chloramphenicol. The secondary structure of the protein variants bound to effectors was measured by Far-UV CD at a ligand concentration of 250 μM. The spectra indicated that the TtgRWT and variant conformations are not altered by the presence of effectors (data not shown). In general, the CD signal monitoring at 222 nm as a function of temperature, showed a significant thermal stabilization upon ligand binding (Table 2). Equivalent thermal unfolding experiments using DSC also revealed that TtgRWT and its mutants are more stable when complexed with the effectors (Table 2, Fig 4). In DSC experiments, the rise of Tm (effector free < Chlr < Nar < Phlr) was concomitant with the increase of the unfolding enthalpic contribution to the protein stability (Table 2) as shown by the enlarged area under the peak, which can be interpreted as an indication of the protein stabilization. Phloretin is the most stabilizing effector, closely followed by naringenin, while the less stabilizing effector is chloramphenicol. The highest Tm was measured for the N110A variant and its thermal stability was less affected by the binding. In the presence of any effector, the less stable variant is H114A. Particularly, the stability of E78A is most dependent on effector binding, being more stable when bound to naringenin and less stable bound to phloretin.


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)

Thermal denaturation followed by DSC of TtgRWT and variants (TtgRH114A, TtgRN110A, TtgRE78A and TtgRS77A) 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).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138469.g004: Thermal denaturation followed by DSC of TtgRWT and variants (TtgRH114A, TtgRN110A, TtgRE78A and TtgRS77A) 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).
Mentions: To characterize the binding of the effectors to TtgRWT and its variants as well as to describe the extent of the effect of each ligand on the structure and stability of the proteins, we performed temperature induced unfolding experiments on all of the variants using DSC and CD in the presence of naringenin, phloretin and chloramphenicol. The secondary structure of the protein variants bound to effectors was measured by Far-UV CD at a ligand concentration of 250 μM. The spectra indicated that the TtgRWT and variant conformations are not altered by the presence of effectors (data not shown). In general, the CD signal monitoring at 222 nm as a function of temperature, showed a significant thermal stabilization upon ligand binding (Table 2). Equivalent thermal unfolding experiments using DSC also revealed that TtgRWT and its mutants are more stable when complexed with the effectors (Table 2, Fig 4). In DSC experiments, the rise of Tm (effector free < Chlr < Nar < Phlr) was concomitant with the increase of the unfolding enthalpic contribution to the protein stability (Table 2) as shown by the enlarged area under the peak, which can be interpreted as an indication of the protein stabilization. Phloretin is the most stabilizing effector, closely followed by naringenin, while the less stabilizing effector is chloramphenicol. The highest Tm was measured for the N110A variant and its thermal stability was less affected by the binding. In the presence of any effector, the less stable variant is H114A. Particularly, the stability of E78A is most dependent on effector binding, being more stable when bound to naringenin and less stable bound to phloretin.

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