Different modes of binding of mono- and biaromatic effectors to the transcriptional regulator TTGV: role in differential derepression from its cognate operator.
Members of the IclR family of regulators exhibit a highly conserved effector recognition domain and interact with a limited number of effectors. In contrast with most IclR family members, TtgV, the transcriptional repressor of the TtgGHI efflux pump, exhibits multidrug recognition properties. A three-dimensional model of the effector domain of TtgV was generated based on the available three-dimensional structure of several IclR members, and a series of point mutants was created. Using isothermal titration calorimetry, we determined the binding parameters of the most efficient effectors for TtgV and its mutant variants. All mutants bound biaromatic compounds with higher affinity than the wild-type protein, whereas monoaromatic compounds were bound with lower affinity. This tendency was particularly pronounced for mutants F134A and H200A. TtgVF134A bound 4-nitrotoluene with an affinity 13-fold lower than that of TtgV (17.4+/-0.6 microM). This mutant bound 1-naphthol with an affinity of 5.7 microM, which is seven times as great as that of TtgV (40 microM). The TtgVV223A mutant bound to DNA with the same affinity as the wild-type TtgV protein, but it remained bound to the target operator in the presence of effectors, suggesting that Val-223 could be part of an intra-TtgV signal recognition pathway. Thermodynamic analyses of the binding of effectors to TtgV and to its mutants in complex with their target DNA revealed that the binding of biaromatic compounds resulted in a more efficient release of the repressor protein than the binding of monoaromatics. The physiological significance of these findings is discussed.
Guazzaroni
María-Eugenia
ME
Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Professor Albareda, 1, E-18008 Granada, Spain.
Gallegos
María-Trinidad
MT
Ramos
Juan L
JL
Krell
Tino
T
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009875:Operator Regions, Genetic; D011084:Polycyclic Aromatic Hydrocarbons / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D016958:Pseudomonas putida / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D026901:Membrane Transport Proteins; D011084:Polycyclic Aromatic Hydrocarbons; D014157:Transcription Factors