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Papers In Press, published online ahead of print January 5, 2006
Biochemistry, Estación Experimental del Zaidín (CSIC), Granada, Granada E18008
Corresponding Author: maritrini.gallegos{at}eez.csic.es
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
J. Biol. Chem, 10.1074/jbc.M511095200
Submitted on October 12, 2005
Revised on January 5, 2006
Accepted on January 5, 2006
Effector-repressor interactions. Binding of a single effector molecule to the operator bound TtgR homodimer mediates derepression
M, 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 (KD of around 1 M), 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 neither 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.
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