title: Molecular Binding Mechanism of TtgR Repressor to Antibiotics and Antimicrobials.
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.
authors.affiliation: Environmental Protection Department, Estación Experimental del Zaidín (EEZ), Spanish National Research Council (CSIC), C/ Profesor Albareda, 1, E-18008 Granada, Spain.
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Fernandez-Escamilla
authors.affiliation: Institute of Molecular and Cellular Biology (IBMC), Miguel Hernández University, Avda/ de la Universidad s/n, E-03202 Elche, Alicante, Spain.
authors.forename: Gregorio
authors.initials: G
authors.lastname: Fernandez-Ballester
authors.affiliation: Physical-Chemistry Department, Faculty of Sciences, University of Granada, Campus Fuentenueva, E-18071 Granada, Spain.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Morel
authors.affiliation: Physical-Chemistry Department, Faculty of Sciences, University of Granada, Campus Fuentenueva, E-18071 Granada, Spain.
authors.forename: Salvador
authors.initials: S
authors.lastname: Casares-Atienza
authors.affiliation: Environmental Protection Department, Estación Experimental del Zaidín (EEZ), Spanish National Research Council (CSIC), C/ Profesor Albareda, 1, E-18008 Granada, Spain.
authors.forename: Juan Luis
authors.initials: JL
authors.lastname: Ramos
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D020125:Mutation, Missense; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D012097:Repressor Proteins; C510127:TtgR protein, Pseudomonas putida