The Multidrug Efflux Regulator TtgV Recognizes a Wide Range of Structurally Different Effectors in Solution and Complexed with Target DNA

• Published in: The Journal of biological chemistry 2005-05, Vol.280 (21), p.20887-20893
• Main Authors: Guazzaroni, María-Eugenia, Krell, Tino, Felipe, Antonia, Ruiz, Raquel, Meng, Cuixiang, Zhang, Xiaodong, Gallegos, María-Trinidad, Ramos, Juan L.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M500783200

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 μm. 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 (KD = 2.4 ± 0.35 nm, ΔH = 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, KD = 4.8 ± 0.19 and 3.0 ± 0.15 μm, respectively. The biological relevance of this finding is discussed.