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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

AuthorsGuazzaroni, María Eugenia CSIC ORCID; Krell, Tino; Felipe, Antonia; Ruiz, Raquel; Meng, Cuixiang; Zhang, Xiaodong; Gallegos, María Trinidad CSIC ORCID ; Ramos, Juan L.
Issue Date13-Mar-2005
PublisherAmerican Society for Biochemistry and Molecular Biology
CitationJournal of Biological Chemistry 280(21): 20887-20893 (2005)
AbstractTtgV 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.
Description7 pages, 4 figures, 4 tables.-- Printed version published May 27, 2005.-- Full-text paper available Open Access at the journal site.
Publisher version (URL)http://dx.doi.org/10.1074/jbc.M500783200
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