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J. Biol. Chem., Vol. 280, Issue 21, 20887-20893, May 27, 2005

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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^*

María-Eugenia Guazzaroni, Tino Krell, Antonia Felipe, Raquel Ruiz, Cuixiang Meng, Xiaodong Zhang, María-Trinidad Gallegos, and Juan L. Ramos¶

From the Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda, 1, E-18008 Granada, Spain and the Centre for Structural Biology, Department of Biological Sciences, Imperial College London, Flowers Building, South Kensington, London, SW7 2AZ, United Kingdom

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 (K_D = 2.4 ± 0.35 nM, H = 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 µM, respectively. The biological relevance of this finding is discussed.

Received for publication, January 21, 2005 , and in revised form, March 9, 2005.

^* This work was supported by EC Project QLK3-CT-2001-0435 (to J. L. R.) and Grant RGY0021/2002 from the Human Frontier Science Program (to M. T. G. and X. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 34-958181600 (ext. 204); Fax: 34-958135740; E-mail: jlramos{at}eez.csic.es.

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