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Please use this identifier to cite or link to this item: http://hdl.handle.net/10481/38654

Title: Molecular Binding Mechanism of TtgR Repressor to Antibiotics and Antimicrobials
Authors: Fernández-Escamilla, Ana María
Fernández-Ballester, Gregorio
Morel, Bertrand
Casares Atienza, Salvador
Ramos Martín, Juan Luis
Issue Date: 2015
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.
Sponsorship: This work was supported by Spanish Ministry of Economy and Competitiveness, National programme for Recruitment and Incorporation of Human Resources, Subprogramme: Ramon y Cajal RYC-2009-04570 and grant P11-CVI-7391 from Junta de Andalucía and EFDR (European Regional Development Fund).
Publisher: Public Library of Science (PLOS)
Keywords: Chloramphenicol
Crystal structure
DNA-binding proteins
Free energy
Electrophoretic mobility shift assay
Pseudomonas putida
URI: http://hdl.handle.net/10481/38654
ISSN: 1932-6203
Rights : Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License
Citation: Fernández-Escamilla, A.M.; et al. Molecular Binding Mechanism of TtgR Repressor to Antibiotics and Antimicrobials. Plos One, 10(9): e0138469 (2015). [http://hdl.handle.net/10481/38654]
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