title: Effector-repressor interactions, binding of a single effector molecule to the operator-bound TtgR homodimer mediates derepression.
abstract: The RND family transporter TtgABC and its cognate repressor TtgR from Pseudomonas putida DOT-T1E were both shown to possess multidrug recognition properties. Structurally unrelated molecules such as chloramphenicol, butyl paraben, 1,3-dihydroxynaphthalene, and several flavonoids are substrates of TtgABC and activate pump expression by binding to the TtgR-operator complex. Isothermal titration calorimetry was employed to determine the thermodynamic parameters for the binding of these molecules to TtgR. Dissociation constants were in the range from 1 to 150 microm, the binding stoichiometry was one effector molecule per dimer of TtgR, and the process was driven by favorable enthalpy changes. Although TtgR exhibits a large multidrug binding profile, the plant-derived compounds phloretin and quercetin were shown to bind with the highest affinity (K(D) of around 1 microm), in contrast to other effectors (chloramphenicol and aromatic solvents) for which exhibited a more reduced affinity. Structure-function studies of effectors indicate that the presence of aromatic rings as well as hydroxyl groups are determinants for TtgR binding. The binding of TtgR to its operator DNA does not alter the protein effector profile nor the effector binding stoichiometry. Moreover, we demonstrate here for the first time that the binding of a single effector molecule to the DNA-bound TtgR homodimer induces the dissociation of the repressor-operator complex. This provides important insight into the molecular mechanism of effector-mediated derepression.
authors.affiliation: Department of Biochemistry, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Apartado de Correos 419, E-18008 Granada, Spain.
authors.forename: Wilson
authors.initials: W
authors.lastname: Terán
authors.forename: Tino
authors.initials: T
authors.lastname: Krell
authors.forename: Juan Luis
authors.initials: JL
authors.lastname: Ramos
authors.forename: María-Trinidad
authors.initials: MT
authors.lastname: Gallegos
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D002151:Calorimetry; D002701:Chloramphenicol / Q000494:pharmacology; D019281:Dimerization; D018432:Drug Resistance, Multiple; D019277:Entropy; D019143:Evolution, Molecular; D005419:Flavonoids / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D006358:Hot Temperature; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009284:Naphthols / Q000494:pharmacology; D009875:Operator Regions, Genetic; D010226:Parabens / Q000494:pharmacology; D010693:Phloretin / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000378:metabolism*; D011794:Quercetin / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D012997:Solvents / Q000737:chemistry; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D005419:Flavonoids; D009284:Naphthols; D010226:Parabens; D012097:Repressor Proteins; D012997:Solvents; C510127:TtgR protein, Pseudomonas putida; C038091:butylparaben; C027029:naphthoresorcinol; D002701:Chloramphenicol; D011794:Quercetin; D001616:beta-Galactosidase; D010693:Phloretin