PMID:15767250

Citation	Guazzaroni, ME, Krell, T, Felipe, A, Ruiz, R, Meng, C, Zhang, X, Gallegos, MT and Ramos, JL (2005) The multidrug efflux regulator TtgV recognizes a wide range of structurally different effectors in solution and complexed with target DNA: evidence from isothermal titration calorimetry. J. Biol. Chem. 280:20887-93
Abstract	TtgV modulates the expression of the ttgGHI operon, which encodes an efflux pump that extrudes a wide variety of chemicals including mono- and binuclear aromatic hydrocarbons, aliphatic alcohols, and antibiotics of dissimilar chemical structure. Using a 'lacZ fusion to the ttgG promoter, we show that the most efficient in vivo inducers were 1-naphthol, 2,3-dihydroxynaphthalene, 4-nitrotoluene, benzonitrile, and indole. The thermodynamic parameters for the binding of different effector molecules to purified TtgV were determined by isothermal titration calorimetry. For the majority of effectors, the interaction was enthalpy-driven and counterbalance by unfavorable entropy changes. The TtgV-effector dissociation constants were found to vary between 2 and 890 mum. There was a relationship between TtgV affinity for the different effectors and their potential to induce gene expression in vivo, indicating that the effector binding constant is a major determinant for efficient efflux pump gene expression. Equilibrium dialysis and isothermal titration calorimetry studies indicated that a TtgV dimer binds one effector molecule. No evidence for the simultaneous binding of multiple effectors to TtgV was obtained. The binding of TtgV to a 63-bp DNA fragment containing its cognate operator was tight and entropy-driven (K(D) = 2.4 +/- 0.35 nm, DeltaH = 5.5 +/- 0.04 kcal/mol). The TtgV-DNA complex was shown to bind 1-napthol with an affinity comparable with the free soluble TtgV protein, K(D) = 4.8 +/- 0.19 and 3.0 +/- 0.15 mum, respectively. The biological relevance of this finding is discussed.
Links	PubMed Online version:10.1074/jbc.M500783200
Keywords	Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Calorimetry/methods; DNA, Bacterial/metabolism; Escherichia coli/genetics; Gene Expression Regulation, Bacterial/drug effects; Indoles/metabolism; Indoles/pharmacology; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Naphthols/metabolism; Naphthols/pharmacology; Nitriles/metabolism; Nitriles/pharmacology; Operon/genetics; Promoter Regions, Genetic/genetics; Protein Binding; Pseudomonas putida/chemistry; Pseudomonas putida/genetics; Recombinant Fusion Proteins; Thermodynamics; Toluene/analogs & derivatives; Toluene/metabolism; Toluene/pharmacology; Transcription, Genetic; beta-Galactosidase/genetics
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