The Multidrug Efflux Regulator TtgV Recognizes a Wide Range of Structurally Different Effectors in Solution and Complexed with Target DNA
2005; Elsevier BV; Volume: 280; Issue: 21 Linguagem: Inglês
10.1074/jbc.m500783200
ISSN1083-351X
AutoresMarı́a-Eugenia Guazzaroni, Tino Krell, Antonia Felipe, Raquel Ruiz‐García, Cuixiang Meng, Xiaodong Zhang, María‐Trinidad Gallegos, Juan L. Ramos,
Tópico(s)Antibiotic Resistance in Bacteria
ResumoTtgV 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. 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. Pseudomonas putida DOT-T1E is a paradigm of solvent-tolerant microorganisms because it can grow in the presence of high concentrations of extremely toxic and harmful compounds such as aromatic hydrocarbons (1Ramos J.L. Duque E. Gallegos M.T. Godoy P. Ramos-González M.I. Rojas A. Terán W. Segura A. Annu. Rev. Microbiol. 2002; 56: 743-768Crossref PubMed Scopus (633) Google Scholar, 2Ramos J.L. Duque E. Huertas M.J. Haïdour A. J. Bacteriol. 1995; 177: 3911-3916Crossref PubMed Google Scholar). These compounds preferentially partition in the cell membrane, disorganizing it and leading to cell death (3Sikkema J. de Bont J.A.M. Poolman B. Microbiol. Rev. 1995; 59: 201-222Crossref PubMed Google Scholar). Efflux pumps have been shown to play a critical role in the removal of toxic compounds such as antibiotics, biocides, dyes, detergents, fatty acids, and organic solvents from the cell membranes (2Ramos J.L. Duque E. Huertas M.J. Haïdour A. J. Bacteriol. 1995; 177: 3911-3916Crossref PubMed Google Scholar, 4Chatterjee A. Chaudhuri S. Saha G. Gupta S. Chowdhury R. J. Bacteriol. 2004; 186: 6809-6814Crossref PubMed Scopus (46) Google Scholar, 5Lee E.H. Rouquette-Loughlin C. Folster J.P. Shafer W.M. J. Bacteriol. 2003; 185: 7145-7152Crossref PubMed Scopus (61) Google Scholar, 6Lee E.W. Huda M.N. Kuroda T. Mizushima T. Tsuchiya T. Antimicrob. Agents Chem. 2003; 47: 3733-3738Crossref PubMed Scopus (118) Google Scholar, 7Middlemiss J.K. Poole K. J. Bacteriol. 2004; 186: 1258-1269Crossref PubMed Scopus (66) Google Scholar, 8Nikaido H. J. Bacteriol. 1996; 178: 5853-5859Crossref PubMed Scopus (855) Google Scholar, 9Ramos J.L. Duque E. Godoy P. Segura A. J. Bacteriol. 1998; 180: 3323-3329Crossref PubMed Google Scholar, 10Schnappinger D. Schubert P. Berens C. Pfleiderer K. Hillen W. J. Biol. Chem. 1999; 274: 6405-6410Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 11van Dyk T. Templeton L.J. Cantera K.A. Sharpe P.L. Sariaslani F.S. J. Bacteriol. 2004; 186: 7196-7204Crossref PubMed Scopus (99) Google Scholar, 12Yoshida K.I. Ohki Y.H. Murata M. Kinehara M. Matsouka H. Satomura T. Ohki R. Kumano M. Yamane K. Fujita Y. J. Bacteriol. 2004; 186: 5640-5648Crossref PubMed Scopus (19) Google Scholar, 13Yu E.W. Aires J.R. Nikaido H. J. Bacteriol. 2003; 185: 5657-5664Crossref PubMed Scopus (148) Google Scholar). In P. putida DOT-T1E, the cooperative action of up to three efflux pumps, TtgABC, TtgDEF, and TtgGHI, is needed to achieve maximal tolerance against toluene, one of the most toxic aromatic hydrocarbons. TtgGHI appears to be the most important extrusion element since, in contrast to the other two efflux pumps, a knock-out mutant in which this efflux pump is not functional was not able to withstand a sudden 0.3% (v/v) toluene shock regardless of the growth conditions (14Rojas A. Duque E. Mosqueda G. Golden G. Hurtado A. Ramos J.L. Segura A. J. Bacteriol. 2001; 183: 3967-3973Crossref PubMed Scopus (205) Google Scholar). TtgGHI, like other multidrug-resistant pumps, possesses a broad substrate specificity reflected in its capacity to extrude not only aromatic hydrocarbons such as toluene, xylenes, or styrene but also aliphatic alcohols such as octanol, nonanol, and decanol, as well as antibiotics of different chemical structure, e.g. ampicillin, tetracycline, and nalidixic acid (14Rojas A. Duque E. Mosqueda G. Golden G. Hurtado A. Ramos J.L. Segura A. J. Bacteriol. 2001; 183: 3967-3973Crossref PubMed Scopus (205) Google Scholar, 15Rojas A. Duque E. Schmid A. Hurtado A. Ramos J.L. Segura A. Appl. Environ. Microbiol. 2004; 70: 3637-3643Crossref PubMed Scopus (55) Google Scholar).The expression of the ttgGHI operon is regulated by the TtgV protein (16Rojas A. Segura A. Guazzaroni M.E. Terán W. Hurtado A. Gallegos M.T. Ramos J.L. J. Bacteriol. 2003; 185: 4755-4763Crossref PubMed Scopus (64) Google Scholar). The ttgV gene is transcribed divergently from the ttgGHI operon, and the corresponding promoters, called PttgV and PttgG, overlap each other. The two start codons are separated by only 210 bp, 40 bp of which constitute the TtgV operator so that TtgV covers the -10 region of ttgG promoter and the -35 region of ttgV promoter (16Rojas A. Segura A. Guazzaroni M.E. Terán W. Hurtado A. Gallegos M.T. Ramos J.L. J. Bacteriol. 2003; 185: 4755-4763Crossref PubMed Scopus (64) Google Scholar, 17Guazzaroni M.E. Terán W. Zhang X. Gallegos M.T. Ramos J.L. J. Bacteriol. 2004; 186: 2921-2927Crossref PubMed Scopus (34) Google Scholar). Basal expression from the ttgG and ttgV promoters occurs, but expression has been shown to increase in response to the presence of some, but not all, of the pump substrates in the culture medium. Direct evidence of in vitro TtgV binding to drugs has only been obtained with 1-hexanol; Guazzaroni et al. (17Guazzaroni M.E. Terán W. Zhang X. Gallegos M.T. Ramos J.L. J. Bacteriol. 2004; 186: 2921-2927Crossref PubMed Scopus (34) Google Scholar) showed in EMSA 1The abbreviations used are: EMSA, electrophoretic mobility shift assay; ITC, isothermal titration calorimetry; Km, kanamycin; LB, Luria-Bertani culture medium. 1The abbreviations used are: EMSA, electrophoretic mobility shift assay; ITC, isothermal titration calorimetry; Km, kanamycin; LB, Luria-Bertani culture medium. that this aliphatic alcohol released TtgV from its target operator. The present study was undertaken to determine the effector profile of TtgV, elucidate the TtgV-effector stoichiometry, and determine the thermodynamic parameters for the binding of the most potent effectors. Furthermore, the binding of 1-naphthol, one of the most potent effectors, by free TtgV and the protein complexed to its operator DNA has been compared using ITC.EXPERIMENTAL PROCEDURESBacterial Strains, Plasmids, and Culture Medium—The bacterial strains and plasmids used in this study are shown in Table I. Bacterial strains were grown in LB medium at 30 °C as described before (14Rojas A. Duque E. Mosqueda G. Golden G. Hurtado A. Ramos J.L. Segura A. J. Bacteriol. 2001; 183: 3967-3973Crossref PubMed Scopus (205) Google Scholar) or in 2×YT for the production of the TtgV protein (18Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1991Google Scholar). Liquid cultures were shaken on an orbital platform operating at 200 rpm. When required, the following antibiotics were added to the cultures: Km, 50 μg/ml; rifampicin, 20 μg/ml; and tetracycline, 20 μg/ml.Table IStrains and plasmids used in this study Apr, Kmr, Rifr, Tcr, and Tolr stand for resistance to ampicillin, kanamycin, rifampicin, tetracycline and toluene, respectively.Strains and plasmidsCharacteristicsRelevant source or referencesP. putidaDOT-T1ERifr, Tolr2Ramos J.L. Duque E. Huertas M.J. Haïdour A. J. Bacteriol. 1995; 177: 3911-3916Crossref PubMed Google ScholarE. coliB834 (DE3)F-, ompI hsdSB (r- B m- B) gal dem metNovagenpANA96Tcr, ttgG promoter cloned in pMP22016Rojas A. Segura A. Guazzaroni M.E. Terán W. Hurtado A. Gallegos M.T. Ramos J.L. J. Bacteriol. 2003; 185: 4755-4763Crossref PubMed Scopus (64) Google ScholarpET29a(+)Kmr, protein expression vectorNovagenpGG1Apr, pUC18 bearing an 8-kb BamHI fragment with ttgGHI and ttgVW14Rojas A. Duque E. Mosqueda G. Golden G. Hurtado A. Ramos J.L. Segura A. J. Bacteriol. 2001; 183: 3967-3973Crossref PubMed Scopus (205) Google ScholarpMP220Tcr, promoterless lacZ expression vector37Spaink H.P. Okker R.J.H. Wijffelman C.A. Pees E. Lugtenberg B.J.J. Plant Mol. Biol. 1987; 9: 27-39Crossref PubMed Scopus (560) Google ScholarpTE103-PttgGApr, promoter of ttgG cloned upstream of the T7 terminator in pTE10317Guazzaroni M.E. Terán W. Zhang X. Gallegos M.T. Ramos J.L. J. Bacteriol. 2004; 186: 2921-2927Crossref PubMed Scopus (34) Google ScholarpTGF2Kmr, pET29a(+) derivative vector used to produce TtgVThis work Open table in a new tab β-Galactosidase Assays—Plasmid pANA96 carries a transcriptional fusion of the PttgG promoter region to the ′lacZ gene in the low copy pMP220 promoter probe vector. P. putida DOT-T1E (pANA96) was grown overnight on LB medium with tetracycline. Cultures were diluted to an initial OD660 of 0.05 in the same medium supplemented or not with the chemicals under study at 1 mm. These compounds were dissolved in Me2SO when needed (note that the latter did not interfere with the induction assays performed in this study). When cultures reached an OD660 of 0.9–1.0, β-galactosidase activity was determined in triplicate in permeabilized cells (19Gallegos M.T. Marqués S. Ramos J.L. J. Bacteriol. 1996; 178: 2356-2361Crossref PubMed Google Scholar).TtgV Expression and Purification—Plasmid pTGF2 was constructed by cloning a 784-bp NdeI-BamHI fragment bearing the ttgV open reading frame in the Kmr pET29a(+) plasmid (Novagen) digested with the same enzymes to allow the expression of the native TtgV protein. Plasmid pTGF2 was transformed in Escherichia coli B834 (DE3) cells. The cells were grown in two-liter conical flasks containing 500 ml of 2×YT culture medium with 50 μg/ml Km, incubated at 30 °C with shaking, and induced with 1 mm isopropyl β-d-thiogalactopyranoside when the turbidity of the culture was around 0.7. Then cultures were grown at 22 °C for 3 h, and cells were harvested by centrifugation (10 min at 4000 × g). The cell pellet was resuspended in 0.2 m sodium acetate, 50 mm NaCl, 0.1 mm EDTA, 2 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride and lysed by sonication. After centrifugation at 13,000 × g for 40 min, the supernatant was loaded onto an S-cation column (16/10, Amersham Biosciences) and eluted with a sodium chloride gradient. The fraction containing TtgV was then dialyzed against buffer containing 10 mm Tris-HCl, pH 8.0, 5% (v/v) glycerol, 100 mm NaCl, 0.1 mm EDTA, and 2 mm dithiothreitol, concentrated to 2 ml and loaded onto a Superdex 200 16/20 column (Amersham Biosciences) for gel filtration. The proteins were then concentrated to 4 mg/ml as determined by the Bio-Rad protein assay kit.Electrophoresis Mobility Shift Assay—A 228-bp DNA fragment containing the wild-type PttgG promoter was amplified by PCR from pGG1 using appropriate primers, isolated from agarose gels, and end-labeled with 32P as described before (18Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1991Google Scholar, 20Terán W. Felipe A. Segura A. Rojas A. Ramos J.L. Gallegos M.T. Antimicrob. Agents Chemother. 2003; 47: 3067-3072Crossref PubMed Scopus (105) Google Scholar). About 1 nm labeled DNA (∼1.5 × 104 cpm) was incubated with the indicated amounts of purified TtgV for 10 min at 30 °C in 10 μl of TAPS binding buffer (50 mm Tris-acetate, pH 8.0; 100 mm potassium acetate; 8 mm magnesium acetate; 27 mm ammonium acetate; 3.5% (w/v) polyethylene glycol, and 1 mm dithiothreitol) containing 20 μg/ml poly(dI-dC) and 200 μg/ml bovine serum albumin. Electrophoresis in nondenaturing polyacrylamide gels and analyses were as described before (17Guazzaroni M.E. Terán W. Zhang X. Gallegos M.T. Ramos J.L. J. Bacteriol. 2004; 186: 2921-2927Crossref PubMed Scopus (34) Google Scholar).Single-round in Vitro Transcription Assays with Supercoiled Plasmid DNA—Reactions (20 μl) were performed in STA buffer (25 mm Tris-acetate, pH 8.0, 8 mm magnesium acetate, 3.5% (w/v) polyethylene glycol, 10 mm KCl) containing 100 nm σ70-holoenzyme (Epicenter), 20 units of RNAsin (Promega), 0.1 mm GTP, and 10 nm supercoiled pTE103-PttgG DNA template (10Schnappinger D. Schubert P. Berens C. Pfleiderer K. Hillen W. J. Biol. Chem. 1999; 274: 6405-6410Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The reactions were incubated for 20 min at 30 °C before the addition of the following elongation mixture: 0.1 mm each of ATP, CTP, and UTP; 0.3 μCi of [α-32P]UTP (20 μCi/μl); and 100 μg/ml heparin. After incubation for a further 10 min at 30 °C, the reactions were stopped by chilling to 4 °C, and the product was precipitated with 0.25 volumes of 10 m ammonium acetate and 2.5 volumes of ethanol. The pellets were washed with 80% (v/v) ethanol. Dried pellets were resuspended in 8 μl of water and 4 μl of formamide sequencing dye. Samples were submitted to electrophoresis using a 6.5% (w/v) polyacrylamide denaturing sequencing gel. The results were analyzed using Personal FX equipment software (Bio-Rad).Isothermal Titration Calorimetry—Measurements were performed on a VP-Microcalorimeter (MicroCal, Northampton, MA) at 30 °C. The protein was thoroughly dialyzed against 25 mm Tris acetate, pH 8.0, 8 mm magnesium acetate, 10 mm KCl, and 1 mm dithiothreitol. The protein concentration was determined using the Bradford assay. Stock solutions of 1-naphthol, 2,3-dihydroxynaphthalene, indole, and 4-nitrotoluene at a concentration of 500 mm were prepared in Me2SO and subsequently diluted with dialysis buffer to a final concentration of 0.3 mm (1-naphthol and 2,3-dihydroxynaphthalene), 1.5 mm (indole), and 1 mm (4-nitrotoluene). The appropriate amount of Me2SO (0.1%) was added to the protein sample in each assay. Solutions of benzonitrile (4 mm) and hexanol and toluene (5 mm) were directly prepared in dialysis buffer. All chemicals were manipulated in glass vessels, and effector samples were neither degassed nor filtered, to avoid evaporation or nonspecific binding. Each titration involved a single 2-μl injection and a series of 4-μl injections of effector molecules into the protein solution. For DNA binding studies, oligonucleotides corresponding to both strands of the TtgV operator were synthesized (5′-GGAATTCTCAAGAGTATCACATAATGCTACACTCTACCGCATTACGATTCAGCAACTGCAGAA-3′ and its corresponding complementary oligonucleotide). Annealing was carried out by mixing equimolar amounts (at a concentration of 60 μm) of each complementary oligonucleotide in 0.5 mm Tris-HCl, pH 8.0, 0.5 mm MgCl2. The mixture was incubated 95 °C for 5 min and then chilled on ice and dialyzed in the buffer used for ITC studies. The mean enthalpies measured from injection of the ligand in the buffer were subtracted from raw titration data before data analysis with ORIGIN software (MicroCal). Titration curves were fitted by a nonlinear least squares method to a function for the binding of a ligand to a macromolecule (21Wiseman T. Williston S. Brandts J.F. Lin L.N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2413) Google Scholar). From the curve thus fitted, the parameters ΔH (reaction enthalpy), KA (binding constant, KA = 1/KD), and n (reaction stoichiometry) were determined. From the values of KA and ΔH, the change in free energy (ΔG) and in entropy (ΔS) were calculated with the equation: ΔG = -RT lnKA = Δ - TΔS, where R is the universal molar gas constant and T is the absolute temperature.Measurements of TtgV/Ligand Binding Ratio Using Equilibrium Dialysis Assays—Four samples of TtgV with different protein concentrations were dialyzed against protein buffer containing effectors using Slide-a-Lyzer (Pierce) equipment for 5 days at 4 °C with stirring to ensure equilibrium. The proteins inside the cassette were then denatured by incubating at 100 °C for 5 min to release the bound effectors into the buffer. The denatured proteins were then centrifuged for 2 min at 13,000 × g. Ultraviolet light absorption at the appropriate wavelength was then measured in the supernatants that contained the effectors, and the effector concentrations were determined using the corresponding ϵ extinction coefficients. The concentration of protein-bound effectors was obtained after correction for the effector concentration in the buffer. Protein concentrations were determined with the Bradford assay (Pierce). The binding ratio and dissociation constant were obtained with the following equation: binding ratio = [protein bound]/[effectors bound], KD = [effectors free][protein free]/[effectors bound].RESULTSIn Vivo Effector Profile of TtgV—As an initial approach to the identification of the effectors recognized by TtgV, we used a PttgG::′lacZ fusion (pANA96) to measure β-galactosidase activity in P. putida DOT-T1E cells grown in the absence or in the presence of 1 mm compounds (Fig. 1). The basal level of expression from the ttgG promoter was 438 ± 34 Miller units, and expression increased up to 5-fold in response to 1 mm tested chemicals. The effectors that yielded the highest induction levels (4–5-fold increase) were two-ring aromatic compounds such as 1-naphthol, 2,3-dihydroxynaphthalene, and indole and one-ring aromatic compounds such as benzonitrile and 4-nitrotoluene (Fig. 1). Other compounds such as alkylphenols, halogenated aromatic rings, and aliphatic and aromatic alcohols also behaved as effectors and increased expression from the PttgG promoter by at least 2-fold.In Vitro TtgV-Effector Interactions—Equilibrium dialysis experiments were carried out to shed light into the effector-TtgV stoichiometry. The results for the binding of 1-naphthol and 2,3-dihydroxynaphthalene to TtgV, as shown in Tables II and III, indicated that one effector molecule binds to the TtgV dimer. The apparent affinity for these molecules was in the low micromolar range. The thermodynamic parameters for the interaction of these two effectors as well as those of other effectors (biaromatic, monoaromatic compounds, and aliphatic alcohols) were subsequently determined by ITC at 30 °C. These effectors were chosen to cover the spectrum of chemically different compounds that behaved as inducers in vivo.Table IICharacterization of 1-naphthol binding to TtgV determined by equilibrium dialysis TtgV at the different concentrations given below were dialyzed against buffers containing effectors for 5 days at 4 °C with stirring to ensure equilibrium. Other experimental conditions are given in "Experimental Procedures."[1-naphthol-bound][1-naphthol-free][TtgV-bound][TtgV-free]Binding ratio (effector/dimer TtgV)Kdμmμmμmμmμm3.323.63.70.40.892.93.823.34.10.30.931.710.422.911.71.30.892.911.822.612.50.70.941.3average0.91 ± 0.032.2 ± 0.8 Open table in a new tab Table IIICharacterization of 2,3-dihydroxynaphthalene binding to TtgV determined by equilibrium dialysis TtgV at the different concentrations given below were dialyzed against buffers containing effectors for 5 days at 4 °C with stirring to ensure equilibrium. Other experimental conditions are given in "Experimental Procedures."[2,3-dihydroxynaphthalene-bound][2,3-dihydroxynaphthalene-free][TtgV-bound][TtgV-free]Binding ratio (effector/dimer TtgV)Kdμmμmμmμmμm15.919.116.20.30.980.816.718.917.40.80.962.119.819.721.92.20.901.020.419.221.51.10.950.7average0.95 ± 0.031.2 ± 0.6 Open table in a new tab The titration of TtgV with different effectors (1-naphthol, 2,3-dihydroxynaphthalene, benzonitrile, indole, 4-nitrotoluene, toluene, and 1-hexanol) is characterized by exothermal heat changes, giving rise to hyperbolic binding curves. This was exemplified by the titration with 1-naphthol shown in Fig. 2 (left-hand panel). ITC data were analyzed using n fixed at 0.5 (one effector molecule/dimer) determined by equilibrium dialysis experiments (see above), and satisfactory curve fits were obtained. The corresponding thermodynamic parameters are shown in Table IV. For all tested effectors, with the exception of 2,3-dihydroxynaphthalene, the binding was driven by favorable enthalpy changes and counterbalanced by unfavorable entropy changes (Table IV). In contrast, the thermodynamic mode of the binding of 2,3-dihydroxynaphthalene was different from that of the other effectors. Binding gave rise to only very small exothermic heat changes and was thus driven by entropy changes. It should be noted that dissociation constants for the binding of naphthol and 2,3-dihydroxynaphthalene determined by ITC and equilibrium dialysis were very close (3.0 ± 0.15 and 2.2 ± 0.8 μm for 1-naphthol and 2.3 ± 0.42 and 1.2 ± 0.6 μm for 2,3-dihydroxynaphthalene, respectively). These two bi-aromatic compounds are clearly bound by TtgV with the highest affinity, which is also reflected in its superior in vivo efficiency (Fig. 1).Fig. 2Isothermal titration calorimetry data for the binding of 1-naphthol to TtgV (left-hand panel) and to TtgV saturated with a 63-bp DNA fragment containing the operator (right-hand panel). Left panel, heat changes (upper panel) and integrated peak areas (lower panel) for the injection of a single 2-μl and a series of 4-μl aliquots of 300 μm 1-naphthol in a solution of 6.4 μm TtgV (B) and buffer (A). Right panel, heat changes (upper panel) and integrated peak areas (lower panel) for the injection of a single 2-μl and a series of 4-μl aliquots of 300 μm 1-naphthol in a solution of 6.4 μm TtgV saturated with a DNA fragment containing the operator. Data were fitted with ORIGIN using an n value of 0.5 (one effector molecule/dimer). Derived thermodynamic parameters are given in Table IV.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IVThermodynamic parameters derived from the calorimetric titration of TtgV with effector molecules TtgV solutions at 6–8 μm in 25 mm Tris-acetate, 8 mm magnesium acetate, 10 mm KCl, 1 mm dithiothreitol, pH 8.0, were titrated with 0.3–5 mm solutions of effectors. Experiments were carried out at 30 °C. Further assay conditions are given under "Experimental Procedures."EffectorLigandKDKAΔHTΔSΔGμmm-1kcal/molkcal/molkcal/mol1-NaphtholTtgV3.0 ± 0.15(3.4 ± 0.17) × 105-8.6 ± 0.16-1.1 ± 0.15-7.5 ± 0.031-NaphtholTtgV-DNA complex4.8 ± 0.19(2.1 ± 0.08) × 105-5.2 ± 0.082.1 ± 0.09-7.3 ± 0.022,3-DihydroxynaphthaleneTtgV2.3 ± 0.42(4.4 ± 0.80) × 105-0.8 ± 0.066.9 ± 0.10-7.7 ± 0.104-NitrotolueneTtgV16.9 ± 0.43(5.9 ± 0.15) × 104-14.9 ± 0.18-8.5 ± 0.18-6.4 ± 0.01BenzonitrileTtgV51.3 ± 1.26(1.9 ± 0.48) × 104-12.5 ± 0.14-6.7 ± 0.15-5.8 ± 0.02IndoleTtgV67.1 ± 1.58(1.5 ± 0.04) × 104-13.0 ± 0.17-7.3 ± 0.18-5.7 ± 0.01TolueneTtgV118.0 ± 2.64(8.5 ± 0.19) × 103-6.3 ± 0.07-0.9 ± 0.09-5.4 ± 0.011-HexanolTtgV892.8 ± 135(1.1 ± 0.17) × 103-14.9 ± 0.15-10.8 ± 1.56-4.1 ± 0.09 Open table in a new tab The dissociation constants for the different effectors span the micromolar range (Table IV). Although benzonitrile and indole were shown to be efficient effectors in vivo, the affinity of TtgV for these two chemicals was lower (in the range of 50–70 μm) than for 1-naphthol. Substantially lower affinities were determined for toluene (KD = 118 μm) and 1-hexanol (KD = 892 μm), which may account for the relatively modest activity of these effectors in vivo. The nitro substitution of toluene at position 4 resulted in a substantial increase in ΔH (-6.3 to -14.9 kcal/mol) and an 8-fold increase in affinity (Table IV), which is in agreement with 4-nitrotoluene being a more efficient effector than toluene in vivo. This is compatible with a potential direct recognition of the nitro group at position 4 by TtgV since a nitro group at position 2 or 3 resulted in a less efficient effector.Different effectors have been shown to bind to different sites in the large binding pocket of the QacR protein (22Grkovic S. Brown M.H. Schumacher M.A. Brennan R.G. Skurray R.A. J. Bacteriol. 2001; 183: 7102-7109Crossref PubMed Scopus (77) Google Scholar, 23Murray D.S. Schumacher M.A. Brennan R.G. J. Biol. Chem. 2004; 279: 14365-14371Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 24Schumacher M.A. Miller M.C. Brennan R.G. EMBO J. 2004; 23: 2923-2930Crossref PubMed Scopus (105) Google Scholar, 25Schumacher M.A. Miller M.C. Grkovic S. Brown M.H. Skurray R.A. Brennan R.G. Science. 2001; 294: 2158-2163Crossref PubMed Scopus (326) Google Scholar). This raises the question whether the effector binding pocket of TtgV can accommodate different molecules at a time. We carried out a series of sequential ITC experiments that involved the initial saturation of TtgV with a first effector followed by the titration with a second effector. In a first series of experiments, TtgV (6.4 μm) was saturated by the addition of aliquots of 300 μm 1-naphthol. This complex was titrated with 300 μm 2,3-dihydroxynaphthalene. In a second series of experiments, TtgV (6.7 μm) was saturated with 2,3-dihydroxynaphthalene and subsequently titrated with 1 mm benzonitrile. In both cases, heat changes were very small and corresponded to the competition of two effectors to a single site and not to the simultaneous binding of both effectors to the protein with a physiological relevant affinity (data not shown). We were thus unable to provide evidence for the simultaneous binding of multiple effectors to TtgV.ITC Binding Studies of 1-nNaphthol to the TtgV-DNA Complex—As stated above, TtgV exerts its biological function by an up-regulation of gene expression as a result of the effector-mediated dissociation of the regulatory protein from its operator. ITC experiments were carried out to study the interaction of 1-naphthol with the TtgV-DNA complex. Synthetic 63-bp oligonucleotides corresponding to both strands of the operator sequence protected by DNaseI footprint experiments were synthesized and annealed (see "Experimental Procedures") (17Guazzaroni M.E. Terán W. Zhang X. Gallegos M.T. Ramos J.L. J. Bacteriol. 2004; 186: 2921-2927Crossref PubMed Scopus (34) Google Scholar). In a first series of ITC assays, 8.1 μm TtgV was titrated with 10-μl aliquots of 32 μm DNA at 30 °C in STA buffer. Binding was entropy-driven (ΔH = 5.5 ± 0.04 kcal/mol, TΔS = 17.5 ± 0.09 kcal/mol) and very tight (KD = 2.4 ± 0.35 nm). Experiments were designed so that the protein concentration after saturation with DNA corresponded exactly to the protein concentration used for the titration of unliganded protein with 1-naphthol. After saturation, the TtgV-DNA complex was titrated with 1-naphthol in a similar fashion as the titration of the unliganded protein (Fig. 2, right-hand panel). The resulting heat changes corresponded to the binding of the effector to the protein and to the dissociation of the protein from DNA. Peaks were narrow, indicating that operator/TtgV dissociation occurred immediately upon effector binding by TtgV. After titration, the sample was subjected to EMSA, which demonstrated that protein has dissociated quantitatively from its target operator DNA (data not shown). In a control assay, free DNA at the same concentration as in the titration of the DNA-protein complex was titrated with 1-naphthol. Resulting pe
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