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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

Central panel shows a global view of TtgRWT in complex with phloretin (PDB code 2UXI).Top panel shows a zoom on the binding sites of one monomer containing two molecules of phloretin, bound to low- and high-affinity binding sites. Residues 122–154 in alpha-helix conformation have been hidden for clearly show the biding site. Bottom panel shows a third phloretin molecule bound to the low affinity binding site of the second monomer. Discontinuous, yellow lines denote phloretin hydrogen bond formation. In all panels the mutated residues are highlighted.
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pone.0138469.g001: Central panel shows a global view of TtgRWT in complex with phloretin (PDB code 2UXI).Top panel shows a zoom on the binding sites of one monomer containing two molecules of phloretin, bound to low- and high-affinity binding sites. Residues 122–154 in alpha-helix conformation have been hidden for clearly show the biding site. Bottom panel shows a third phloretin molecule bound to the low affinity binding site of the second monomer. Discontinuous, yellow lines denote phloretin hydrogen bond formation. In all panels the mutated residues are highlighted.

Mentions: The TtgABC efflux pump expression takes place in response to the binding of toxic compounds directly to the TtgR dimer in Pseudomonas putida DOT-T1E. These compounds differ in structure one from another. The importance of some TtgR residues in the ligand-protein interaction have previously been reported (e.g. Arg176, whose mutation to Gly reduced the affinity for phloretin) [12]. It has also been shown that several other mutations altered the affinity of TtgR for its operator [13]. These previous studies provide insufficient data to propose a detailed description of the interactions in the active binding pocket upon ligand binding. To determine critical structural interactions in the TtgR binding process, we performed a deeper examination of the crystallographic structures of the TtgR protein in complex with several natural ligands. We then rationally designed TtgR variants mutated in essential residues involved in the binding process and selected three ligands for the study of regulator/effector interactions, including two plant secondary metabolites (naringenin and phloretin), and an antibiotic (chloramphenicol). These ligands have different methods of interaction with the residues located at the most hydrophobic region of the TtgR binding pocket. A single molecule of chloramphenicol and naringenin are bound per protein monomer, these compounds bind at the high affinity site in a very similar position, with the protein-ligand interactions specific for each effector. Remarkably, phloretin is the only organic compound capable to bind, at the same time, to the high and low affinity binding sites [12] (Fig 1). The crystallographic structural analysis of the TtgR complex with phloretin and naringenin stressed the importance of the polar residues Asn110 and His114 located in the lower part of the binding pocket at helix-α6. This part of the vast cavity corresponds to the high affinity area, near to the repressor binding site and is mostly formed by polar residues (Asn110, His114 and Asp172). Upon binding of phloretin or naringenin, Asn110 is positioned very favorably to be coordinated to the amino and dimethylamino effector groups (Fig 1 top panel). In fact, Asn110 has been proposed, using docking analysis, as a ligand-sensor residue [23]. His114 is one of the polar residues at the high affinity site, away from the ligand compared to Asn110, which plays a putative ‘assistant’ role in the correct positioning of the ligand. In addition to the hydrophobic interactions described for the low affinity binding pocket [12], two residues belonging to helix-α4, Ser77 and Glu78, appeared to play a major role, not only in regard to the ligand binding but also to maintain the global structure of the protein (Fig 1 bottom panel). To get more information about the binding site constraints, it is essential to perform a comparative study of WT crystal structure and its complexed forms. TtgRWT crystal structure has not been solved, this is due to the poor diffraction of the crystals obtained in absence of ligands; this finding indicates a more flexible structure. For this reason, we modeled the TtgRWT structure using the crystal coordinates of TtgRH67A whose mutation was reverted to His (see Materials and Methods section). This approximation is based on the absence of appreciable conformational changes between TtgRH67A and TtgRWT upon ligand binding effectors of different nature. Using the structural information described above we aimed to explore the importance of these residues on the binding mechanism; for this, we selected and mutated Ser77, Glu78, Asn110 and His114 residues to Ala.


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)

Central panel shows a global view of TtgRWT in complex with phloretin (PDB code 2UXI).Top panel shows a zoom on the binding sites of one monomer containing two molecules of phloretin, bound to low- and high-affinity binding sites. Residues 122–154 in alpha-helix conformation have been hidden for clearly show the biding site. Bottom panel shows a third phloretin molecule bound to the low affinity binding site of the second monomer. Discontinuous, yellow lines denote phloretin hydrogen bond formation. In all panels the mutated residues are highlighted.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138469.g001: Central panel shows a global view of TtgRWT in complex with phloretin (PDB code 2UXI).Top panel shows a zoom on the binding sites of one monomer containing two molecules of phloretin, bound to low- and high-affinity binding sites. Residues 122–154 in alpha-helix conformation have been hidden for clearly show the biding site. Bottom panel shows a third phloretin molecule bound to the low affinity binding site of the second monomer. Discontinuous, yellow lines denote phloretin hydrogen bond formation. In all panels the mutated residues are highlighted.
Mentions: The TtgABC efflux pump expression takes place in response to the binding of toxic compounds directly to the TtgR dimer in Pseudomonas putida DOT-T1E. These compounds differ in structure one from another. The importance of some TtgR residues in the ligand-protein interaction have previously been reported (e.g. Arg176, whose mutation to Gly reduced the affinity for phloretin) [12]. It has also been shown that several other mutations altered the affinity of TtgR for its operator [13]. These previous studies provide insufficient data to propose a detailed description of the interactions in the active binding pocket upon ligand binding. To determine critical structural interactions in the TtgR binding process, we performed a deeper examination of the crystallographic structures of the TtgR protein in complex with several natural ligands. We then rationally designed TtgR variants mutated in essential residues involved in the binding process and selected three ligands for the study of regulator/effector interactions, including two plant secondary metabolites (naringenin and phloretin), and an antibiotic (chloramphenicol). These ligands have different methods of interaction with the residues located at the most hydrophobic region of the TtgR binding pocket. A single molecule of chloramphenicol and naringenin are bound per protein monomer, these compounds bind at the high affinity site in a very similar position, with the protein-ligand interactions specific for each effector. Remarkably, phloretin is the only organic compound capable to bind, at the same time, to the high and low affinity binding sites [12] (Fig 1). The crystallographic structural analysis of the TtgR complex with phloretin and naringenin stressed the importance of the polar residues Asn110 and His114 located in the lower part of the binding pocket at helix-α6. This part of the vast cavity corresponds to the high affinity area, near to the repressor binding site and is mostly formed by polar residues (Asn110, His114 and Asp172). Upon binding of phloretin or naringenin, Asn110 is positioned very favorably to be coordinated to the amino and dimethylamino effector groups (Fig 1 top panel). In fact, Asn110 has been proposed, using docking analysis, as a ligand-sensor residue [23]. His114 is one of the polar residues at the high affinity site, away from the ligand compared to Asn110, which plays a putative ‘assistant’ role in the correct positioning of the ligand. In addition to the hydrophobic interactions described for the low affinity binding pocket [12], two residues belonging to helix-α4, Ser77 and Glu78, appeared to play a major role, not only in regard to the ligand binding but also to maintain the global structure of the protein (Fig 1 bottom panel). To get more information about the binding site constraints, it is essential to perform a comparative study of WT crystal structure and its complexed forms. TtgRWT crystal structure has not been solved, this is due to the poor diffraction of the crystals obtained in absence of ligands; this finding indicates a more flexible structure. For this reason, we modeled the TtgRWT structure using the crystal coordinates of TtgRH67A whose mutation was reverted to His (see Materials and Methods section). This approximation is based on the absence of appreciable conformational changes between TtgRH67A and TtgRWT upon ligand binding effectors of different nature. Using the structural information described above we aimed to explore the importance of these residues on the binding mechanism; for this, we selected and mutated Ser77, Glu78, Asn110 and His114 residues to Ala.

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