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Originally published In Press as doi:10.1074/jbc.M610032200 on April 6, 2007

J. Biol. Chem., Vol. 282, Issue 22, 16308-16316, June 1, 2007
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Different Modes of Binding of Mono- and Biaromatic Effectors to the Transcriptional Regulator TTGV

ROLE IN DIFFERENTIAL DEREPRESSION FROM ITS COGNATE OPERATOR*

María-Eugenia Guazzaroni, María-Trinidad Gallegos, Juan L. Ramos1, and Tino Krell

From the Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Professor Albareda, 1, E-18008 Granada, Spain

Received for publication, October 26, 2006 , and in revised form, March 27, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 µM). This mutant bound 1-naphthol with an affinity of 5.7 µM, which is seven times as great as that of TtgV (40 µM). 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The DOT-T1E strain of Pseudomonas putida has the extraordinary capacity to withstand, and even grow in, the presence of high concentrations of organic solvents such as an aqueous solution saturated with toluene, a highly toxic compound (1). The main mechanism underlying this resistance lies in the action of three RND (resistance-nodulation-cell Division) efflux pumps, termed TtgABC, TtgDEF, and TtgGHI (pump encoded by toluene tolerance genes ttgGHI) (2), which extrude organic solvents and other toxic compounds from the cells. These three efflux pumps show a high degree of similarity to the AcrAB-TolC multidrug efflux pump, which is the best characterized member of this family (3–7, 29). Expression of the P. putida efflux pumps is controlled by transcriptional repressors; TtgR controls the expression of the ttgABC operon (8, 9), whereas TtgV (10–12) is the main regulator controlling the expression of the ttgDEF and ttgGHI operons.

TtgV, a member of the IclR family of regulators (13, 14), exhibits multidrug binding properties (12) in contrast to other members of this family, which are generally characterized by their high specificity for effector molecules (15–17). TtgV is a repressor that operates according to effector-mediated derepression. In the absence of effector, the protein is bound to the promotor DNA region repressing transcription. Effector binding to the TtgV-DNA complex is thought to produce an intramolecular signal that is transmitted to the DNA-binding domain, giving rise to protein dissociation from the operator. The RNA polymerase then accesses the promoter and transcribes the corresponding genes (11). The most efficient effectors in vivo are two-ring aromatic compounds such as 1-naphthol (1NL)2 and indole (IND) and one-ring compounds such as 4-nitrotoluene (4NT) and benzonitrile (BN) (12).

In the framework of structural genomic studies, two research groups reported the three-dimensional structure of two members of the IclR family members, that of the IclR-TM protein isolated from Thermotoga maritima (PDB: 1MKM (18)) and that of a regulator of unknown function purified from Rhodococcus sp. RHA1 (PDB: 2G7U). Both proteins consist of two well separated domains. The N-terminal DNA-binding domain is linked by a long helix to the conserved effector-binding domain. Furthermore, the coordinates of several individual effector-binding domains of IclR family members have been released on the PDB data base. However, none of the structures available forms a complex with a physiologically relevant ligand.

A certain body of information is available on the interaction of multidrug-binding transcriptional regulators with effectors (19, 20). In several cases, affinities do not correlate with the potential of the effectors to release protein in vitro or with their capacity to modulate gene expression in vivo (21, 22). This raises questions concerning the efficiency of effector-induced intramolecular signal transmission, an issue that has rarely been addressed. From structural studies, it appears that the effector structure determines with which set of amino acids the effector interacts in the binding pocket (23), which in turn determines the efficiency of signal transduction (21). It is unknown whether this efficiency can also be modulated by mutations in the regulator binding pocket, which would be of relevance for understanding the evolution of multidrug-binding proteins. Based on the three-dimensional homology model of TtgV, we have identified the potential effector-binding pocket of TtgV. Six site-directed alanine replacement mutants of amino acids located in this pocket were generated and characterized in this study. Isothermal titration calorimetry (ITC) was used to determine the thermodynamic parameters for the binding of four different effectors to the wild type and to all TtgV mutants. Further experiments were aimed at evaluating the impact of the mutations on DNA binding and at characterizing the efficiency of the effector in triggering the release of the bound repressor.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Site-directed Mutagenesis—TtgV mutants, in which amino acid residues at positions 118, 134, 140, 200, 204, and 223 were replaced by alanine and valine (positions 134 and 200), were generated by overlapping PCR mutagenesis (24) using plasmid pANA126 (10) as a source of the ttgV wild-type allele. For each mutant, three PCRs were carried out. The initial two PCRs involved amplifications using the upstream primer (5'-CGCTCCACCGTTCAGAGAAT-3', corresponding to nucleotides 139–158 of ttgV coding sequence) and a mismatch primer covering the segment to be mutated as well as a PCR amplification using the downstream primer (5'-CTTGTCGACGGAGCTCGAAT-3', nucleotides 791–810 of the ttgV coding sequence) and an oligonucleotide complementary to the mismatch primer. The following mismatch primers were used, and the mismatch codon is underlined: I118A, 5-AGACAAAGCGTACGTGCTT-3; F134A, 5'-GGTAGTGGCGCCGATTGGTA-3'; V140F, 5'-GTATTAACTTCCCCGCGCA-3'; V140A, 5'-GTATTAACGCGCCCGCCGCA-3'; H200A, 5'-TGGACGAGGCGATTGATGGC-3'; V204A, 5'-ATTGATGGCGCGTGCTCATT-3'; V223A: 5'-CTCGCGATCGCGATGCCGAG-3'. The resulting overlapping PCR products were annealed, supplemented with upstream and downstream primers, and submitted to the third PCR. For the I118A, F134A, and V160A mutations, the final PCR product was cut with BbvCI and BlnI, which produced a 223-bp fragment that was cloned into pANA126 linearized with the same enzymes. For the H200A, H200V, V204A, and V223A mutations, the final PCR product was digested with BlnI and PstI, and the resulting 200-bp fragment was equally cloned into pANA126.

Cell Culture and Protein Expression—Escherichia coli B834 (DE3) was transformed with pANA126 bearing the wild-type ttgV allele (10) and a series of pANA126 derivatives that encode the six different TtgV mutants. Cells were grown in 2-liter conical flasks with 500 ml of LB supplemented with 25 µg/ml kanamycin. Cultures were incubated at 30 °C with shaking and induced with 0.1 mM isopropyl beta-D-thiogalactopyranoside when the culture reached a turbidity at 660 nm (OD660) of 0.7. Cultures were then transferred at 18 °C, and after growth for 3 h, cells were harvested by centrifugation (10 min at 4000 g) and stored at –80 °C.

Protein Purification—Cells from a 1-liter culture were suspended in 50 ml of buffer A (25 mM NaH2PO4/Na2HPO4, 0.5 M NaCl, 10 mM imidazole, 5% (v/v) glycerol, 0.1 mM dithiothreitol, pH 7.5) containing 10 units/ml Benzonase (Novagen, Madrid, Spain) and one tablet of Roche Applied Science CompleteTM EDTA-free protease inhibitor mixture. Cells were broken by two passages through a French press at 1000 p.s.i., and the resulting suspension was centrifuged at 19,000 x g for 45 min. The supernatant was filtered and loaded onto a 5-ml Hi-Trap chelating column (GE Healthcare, St. Gibes, UK). His6-TtgV and its mutant variants were eluted with a 45–500 mM gradient of imidazole in buffer A. Protein was dialyzed against 20 mM Tris-HCl, 8 mM magnesium acetate, 300 mM NaCl, pH 7.2. For storage at –80 °C, the samples were mixed with 10% (v/v) glycerol. The purity of the protein was between 90 and 95%, as judged from SDS-PAGE gels. Protein samples were aliquoted prior to freezing. All experiments were carried out with a single batch of each protein. Protein aliquots were thawed for immediate use, and excess protein was discarded.

ITC—Measurements were made with a VP-Microcalorimeter (MicroCal, Northampton, MA) at 25 °C. The protein was thoroughly dialyzed against 20 mM Tris-HCl, 8 mM magnesium acetate, 100 mM NaCl, 10% (v/v) glycerol, and 1 mM dithiothreitol, pH 7.2. The buffer used in our initial analysis (12) and in the present study differed in that the buffer used for the assays reported here included 10% (v/v) glycerol, which had a stabilizing effect on the protein, as well as a higher ionic strength and lower pH (7.2 rather than 8.0), which corresponds more closely to physiological conditions. Protein concentration was determined with the Bradford assay. Stock solutions of 1NL, BN, 4NT, and IND at a concentration of 500 mM were prepared in dimethyl sulfoxide, and the solutions were diluted with dialysis buffer to final concentrations of 0.5 to 1 mM. The corresponding amount of dimethyl sulfoxide was added to the protein sample. DNA duplex samples were prepared as described by Guazzaroni et al. (12). Each titration involved a single 1.6-µl injection and a series of 4.8-µl injections of effectors into a 34–40 µM protein solution. The mean enthalpies measured from injection of the ligands into the buffer were subtracted from raw titration data prior to data analysis with a model for the binding of a ligand to identical independent sites of a macromolecule (MicroCal). Data analysis with this model produced satisfactory statistical data.

Circular Dichroism—The CD spectra of each protein were recorded on a Jasco 715 spectropolarimeter (Great Dunmow, UK). Spectra in the far UV region (195–260 nm) were recorded in cylindrical quartz cells (0.02-cm path length) at a protein concentration of 0.6 mg/ml. All protein solutions were dialyzed against the buffer used for ITC.


Figure 1
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  		FIGURE 1.
The three-dimensional model of the effector-binding domain of TtgV. A, a surface plot colored according to electrostatic potential. The assumed ligand-binding pocket is highlighted, and the amino acids replaced by alanine are shown in ball-and-stick mode. B, ribbon representation of the model, annotated to show secondary structure elements (H for helix, S for strand) and the mutated amino acids. The figures were prepared with WebLabViewer software (Accelrys, San Diego, CA).

 
EMSA—Experiments were carried out as described previously (12). One nM labeled DNA probe (~1.5 x 104 cpm) was incubated with increasing concentration of TtgV (10–1500 nM) for 10 min at 30 °C in 10 µl of 20 mM Tris-HCl, 8 mM magnesium acetate, 100 mM NaCl, 10% (v/v) glycerol, and 1 mM dithiothreitol, pH 7.2, containing 20 µg/ml poly(dI-dC) and 200 µg/ml bovine serum albumin.

Determination of MICs—P. putida DOT-T1E and isogenic mutants lacking one or several Ttg efflux pumps (2, 25) were grown overnight in LB medium with the appropriate antibiotics. Cultures were diluted 100-fold in LB supplemented with solvents at different concentrations and incubated for 20 h at 30 °C. The MIC value corresponds to the lowest concentration that reduced growth by more than 90%. Results are the average of at least five independent assays.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Homology Modeling of TtgV and Identification of Amino Acids in the Effector-binding Site—We have previously shown that the IclR family of regulators comprises more than 500 members and that a distinct signature profile can be derived from the alignment of the whole set of proteins. In contrast with other families of regulators, the region that best defines the IclR family is not the DNA-binding domain but the effector-binding region (13, 25). The predicted secondary structure of TtgV can be closely aligned to the secondary structure elements found in the three-dimensional structure of the full-length IclR-TM protein of T. maritima (18). The TtgV sequence corresponding to the effector-binding domain was subsequently subjected to homology modeling with Geno3D (26) software. The model was based on the four following templates: T. maritima IclR-TM (PDB: 1MKM (18)), the effector-binding domains of E. coli IclR (PDB: 1TD5), the glyoxylate regulatory protein (PDB: 1TF1), and KdgR (PDB: 1YSP). These templates share 21–29% sequence identity with TtgV. The model obtained was submitted to What_check (27) and was found to have an acceptable geometry. A Ramachandran plot showed that over 97% of the residues were in allowed regions. With the DALI algorithm, it was possible to superimpose the TtgV effector-binding domain model onto its templates with C{alpha} root mean square deviation values between 1.8 and 2.4 Å. A surface representation of this model (Fig. 1A) shows a hydrophobic cavity with a volume of 1200 Å3 as determined by PASS software (28). This model proposes that the ligand-binding pocket of TtgV is formed by a long loop connecting S2 with H3 and the beta-sheet (Fig. 1B). A number of residues within this cavity were selected for mutagenesis according to two criteria: 1) their location and projection in the binding pocket of the model and 2) their conservation in TtgT, a transcriptional regulator sharing 56% sequence identity with TtgV, which has a very similar effector profile.3

Based on these criteria, 6 residues, Ile-118, Phe-134, Val-140, His-200, Val-204, and Val-233, were chosen (Fig. 1B), and alanine replacement mutants were generated in each position. In the model, 2 amino acids were located on beta-strands: Ile-118 on S2 and Val-223 on S4. Residues Phe-134 and Val-140 were located on the loop connecting S2 with H3. The side chain of Phe-134 lies in the lower side of the effector-binding pocket and appears to play a central role in effector binding. Amino acids His-200 and Val-204 were mutated in the upper part of the pocket.

Mutations in the predicted effector-binding pocket seemed not to alter the secondary protein structure of the mutant proteins, as deduced from analysis of mutant and wild-type TtgV proteins by far UV circular dichroism spectroscopy. The spectra of all proteins could be closely superimposed (Fig. 2), indicating that the amino acid replacements did not significantly affect the protein secondary structure.


Figure 2
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  		FIGURE 2.
Circular dichroism analysis of mutant and wild-type TtgV. Superimposed far-UV circular dichroism spectra of TtgV (——), TtgVH200A (····), TtgVI118A (—–), TtgVF134A (–··–··), TtgVV140A (– – –), TtgVV204A (·–·–·–), and TtgVV223A (–||–||–).

 
Most Mutants Show Increased Affinities for Biaromatic Effectors but Bind Monoaromatic Compounds with Lower Affinity—We recently showed that TtgV effectors are primarily monoaromatic and biaromatic compounds (12). Two representatives of each class, which are efficient effectors in vivo, the monoaromatic compounds 4NT and BN and the biaromatic effectors 1NL and IND, were chosen to study their binding parameters to native and mutant proteins using ITC. With this technique, all thermodynamic binding parameters can be determined in a single experiment.


Figure 3
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  		FIGURE 3.
Isothermal titration calorimetry study of the binding of 4NT and 1NL to the TtgV and TtgV F134A mutant. A, upper panel, Raw ITC data, injection of 1.6- and 4.8-µl aliquots of 4NT (1 mM) into 37 µM TtgV and 35 µM F134A mutant (tetramer concentration). For clarity, the traces have been displaced arbitrarily along the x axis. Lower panel, integrated and corrected-for-dilution peak areas. B, microcalorimetric titration of 40 µM TtgV and 37 µM TtgVF134A mutant with 0.5 mM 1NL, using the same injection protocol as in panel A. The symbols are as follows: squares, wild-type TtgV protein; circles, TtgVF134A mutant protein. Thermodynamic parameters are shown in Table 1.

 
Fig. 3A shows the titration of TtgV and the F134A mutant with 4NT. The derived binding parameters are given in Table 1. Binding to the TtgV protein was enthalpy-driven ({Delta}H = –9.7 ± 0.2 kcal/mol) and characterized by a KD of 17.4 ± 0.6 µM. When the same experiment was repeated with the F134A mutant, the heat changes were much smaller, and affinity decreased by a factor of almost 13-fold (Fig. 3A and Table 1). An even more dramatic decrease in affinity for 4NT (27-fold) was obtained for the H200A mutant. For both mutants, enthalpy changes were reduced to –3.0 and –3.5 kcal/mol, respectively (Table 1), which is consistent with fewer molecular interactions between the effector and these mutants. Affinity of the TtgVV140A, TtgVV204A, and TtgVV223A mutants was reduced by one-half to two-thirds of that of the native TtgV protein (Table 1). We also found that the single mutant TtgV I118A showed 2-fold higher affinity for 4NT than the wild-type protein.


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  		TABLE 1
Thermodynamic parameters derived from the microcalorimetric titration of TtgV with one- and two-ring aromatic effectors

Data were derived from ITC experiments; for further details, see "Experimental Procedures." One-ring aromatic compounds were 4-nitrotoluene (4NT) and benzonitrile (BN); two-ring aromatic compounds were 1-naphthol (1NL) and indole (IND). Experiments were carried out in 20 mM Tris-HCl, 8 mM magnesium acetate, 100 mM NaCl, 10% (v/v) glycerol, and 1 mM dithiothreitol, pH 7.2.

 
The tendency in affinity observed for 4NT was completely opposite when the same experiments were repeated with the biaromatic effector 1NL; all mutants showed a higher affinity than the wild-type protein (Table 1). This is illustrated in Fig. 3B for the wild type and the F134A mutant, which bound 1NL with KD values of 40.1 ± 3 and 5.7 ± 0.2 µM, respectively. All six mutants exhibited affinities for 1NL that were between 1.7- and 13.8-fold higher. Most interestingly, the gain in affinity for 1NL was most pronounced for the mutants that had the lowest affinity for the monoaromatic 4NT, i.e. the F134A and H200A mutants.

To establish whether the differential binding behavior of these two effectors is a general phenomenon for mono- and biaromatic effectors, ITC experiments were conducted with two other representative effectors, namely BN (monoaromatic) and IND (biaromatic). In a manner exactly analogous to 4NT, all mutants except I118A had decreased affinity for the monoaromatic compound BN. This reduction in affinity was again particularly pronounced for F134A and H200A (Table 1). In analogy to 4NT, the enthalpic contribution to the binding of BN to F134A was again reduced, and no detectable affinity was observed for the H200A protein. As in the experiments with 1NL, all mutants were found to have increased affinities with respect to IND (Table 1).

We hypothesized that the increase in affinity for bicyclic compounds is founded on the increase in the volume of the binding pocket caused by the replacement of bulky amino acids. To further study this unexpected finding, we generated mutants in which Phe-134 and His-200 were replaced by valine. The microcalorimetric titrations also showed that both mutants bound monocyclic compounds with decreased affinity when compared with the wild-type protein (Table 1). Although F134A and H200V bound bicyclic compounds with higher affinity than the parental wild-type protein, affinity was lower than that of the corresponding alanine replacement mutants. Therefore, these results support the above hypothesis. We have also generated the V140F change. In this case, we found that the decrease in the size of the pocket resulted in small changes in affinity for the 4NT, BN, and IND compounds when compared with the V140A mutant. In contrast, 1NL bound with a higher affinity to V140F than to V140A. This indicates that apart from the volume increase, there are other factors, such as the establishment of additional van der Waals interactions between Val-140 and 1-NL, that can influence effector recognition (3, 5, 20, 21).

We also determined the affinity of the wild-type TtgV and its mutant variants when complexed with DNA for mono- and biaromatic effectors. We found, in agreement with previous findings (12), that affinity of TtgV and TtgV mutants for their effectors when bound to DNA was not altered in a significant manner. To illustrate this, we performed experiments involving the titration of free and DNA-bound TtgVV223A with 1NL (Fig. 4). 1NL bound to free and DNA-bound TtgVV223A with similar affinities, as evidenced by their respective dissociation constants of 6.2 ± 0.4 and 4.8 ± 0.1 µM.

Effect of Mutations on Affinity for Operator DNA—Subsequent experiments were aimed at elucidating whether the mutations in the effector-binding site altered the DNA binding characteristics of the mutants. EMSAs using 1 nM 210-bp fragment corresponding to the entire ttgV-ttgGHI intergenic region were carried out with increasing concentrations of each protein (10–1500 nM). From the fractions of bound and free DNA, the apparent dissociation constants were calculated, which are listed in Table 2. The wild-type TtgV protein had a KDapp of 150 ± 5nM. The TtgVV223A mutant exhibited affinity similar to that of the wild type, whereas the remaining five proteins had lower affinities for their target DNA operator (their affinity decreased by factors of 3.7–5.4). An additional control experiment was done with mutant TtgVC205S. This mutation is vicinal to Val-204, but according to the homology model, it is unlikely to be involved in effector binding. Mutant TtgVC205S was found to have unaltered DNA binding properties when compared with the wild-type protein. This set of results suggested that mutations in the effector-binding pocket had a measurable effect on the DNA binding properties of this regulator, which is consistent with the existence of an intra-TtgV signal transmission chain connecting the two critical domains of this protein. This has been documented with other regulators such as TetR (30, 31), AraC (32, 33), MelR (34), XylS (35, 36), and XylR (37).


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  		TABLE 2
Determination of apparent dissociation constants of wild-type and TtgV mutants for its operator

Data were obtained from the densitometric analyses of EMSA in the presence of protein concentrations ranging from 10 to 1500 nM. Results shown are the means of at least two independent assays done in duplicate. The DNA probe was 1 nM of a 210-bp fragment comprising the entire intergenic ttgV-ttgG region.

 


Figure 4
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  		FIGURE 4.
1NL binding to the TtgV-DNA complex and quantification of the amount of protein released. Upper panel, raw data for the microcalorimetric titration of TtgV, TtgVV223A, and TtgVV223A-DNA complex with 0.5 mM 1NL (initial injection of 1.6 µl followed by 4.8-µl aliquots). In all cases, the protein concentration was 40 µM, and 15 µM 63-bp operator DNA fragment was present in the latter sample. The sequence and preparation of the 63-bp DNA duplex were as described by Guazzaroni et al. (12). For clarity, the traces have been displaced arbitrarily along the y axis. Lower panel, integrated peak areas of the raw titration data and curve fit. The symbols are as follows: {blacksquare}, wild-type TtgV; {square}, TtgVV223A; {circ}, DNA-TtgVV223A.

 


Figure 5
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  		FIGURE 5.
EMSA of the ttgV-ttgGHI intergenic region in the presence of TtgV (A) and TtgVV223A (B) with different effectors. One nM DNA fragment comprising the 210-bp intergenic ttgV-ttgG region was incubated without (first lane) and with 1µM wild-type TtgV (A) or the mutant TtgVV223A (B) in the absence of effectors (–) or with the indicated effectors at concentrations corresponding to 1 or 10 times their KD as determined by ITC (Table 1). B, bound DNA; F, free DNA.

 
Differential Efficiency of Derepression of the Four Effectors—We then reasoned that if an intra-TtgV signal chain exists, it might be possible for different effector molecules to influence the efficiency of transmission, and subsequently, the functioning of TtgV. To test this hypothesis, we conducted EMSA with constant amounts of DNA and TtgV but with effector concentrations corresponding to multiples of the KD. Under these conditions, occupancy of a protein-DNA complex with the effector is constant. Differences in effector-mediated protein release can thus be assumed to reflect differences in the efficiency of intra-TtgV signal transduction rather than differences in affinity.

Fig. 5A shows the EMSA results for TtgV in the presence of 1 and 10 times the KD value determined for each effector. The densitometric analysis of this gel revealed that at a concentration of 1 x KD of 1NL, about 70% of the DNA was freed from the DNA/TtgV complex. However, in the corresponding experiments with 4NT, IND, and BN, the amount of freed DNA was low (between 1 and 3%). At effector concentrations corresponding to 10 x KD, almost complete release of DNA was observed for the two biaromatic effectors 1NL (97%) and IND (92%), whereas the amount of freed DNA in the presence of the monoaromatic compounds 4NT and BN was only 33 and 30%, respectively. These data are consistent with the different efficiency in signal transduction by different effectors, biaromatic compounds being more effective than monoaromatic effectors. Similar assays were conducted with all mutants, and the results were similar to those reported for the wild-type protein (not shown) except for TtgVV223A. EMSAs revealed that this mutant protein was not readily released from its target operator in the presence of effectors, in contrast to the wild-type protein. The experiment illustrated in Fig. 5B shows that with concentrations of 1NL corresponding to 10 times its KD, a large amount (>80% in a series of three independent assays) of the TtgVV223A mutant remained bound to DNA, which is not the case for the wild-type protein (Fig. 5A).

Analogous experiments with TtgVV223A were carried out using the effectors 4NT and BN. Both these effectors bind with weaker affinity to mutant proteins (Table 1), which implies that the concentrations used were superior to those added to the wild-type TtgV. However, in analogy to the experiments with 1NL, the amount of TtgVV223A released by 4NT and BN was almost negligible. These results are consistent with TtgVV223A being less efficient in effector-mediated protein release.

Biaromatic TtgV Effectors Are Substrates for the TtgDEF and TtgGHI Efflux Pumps—To evaluate the physiological relevance of the above data, it appeared indispensable to elucidate whether the effectors tested are substrates of the efflux pumps TtgDEF and TtgGHI. The MICs for these four compounds were determined with the parental P. putida DOT-T1E strain as well as in three mutants lacking the TtgDEF, TtgGHI, or both RND pumps (Table 3). A clear increase in susceptibility to 1NL and IND was seen in the strains that lacked the TtgDEF or TtgGHI pumps, which is direct evidence that these two compounds are substrates of the pumps under study. However, susceptibility to 4NT and BN was only slightly affected. These data are consistent with primarily biaromatic compounds acting as substrates for the TtgDEF and TtgGHI pumps.


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  		TABLE 3
MIC of TtgV effectors for the wild-type P. putida DOT-T1E strain and its isogenic mutant strains

The mutant strains had a knock-out in the genes encoding the indicated efflux pumps (2, 25). Data are the average and standard deviation of at least three independent assays.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Localization of the Effector-binding Site—A homology model of the effector-binding domain of TtgV was generated, and 6 amino acids located in an apparent binding pocket were mutated. All mutants had altered binding parameters for the four effectors used (Fig. 3 and Table 1). Recently, the three-dimensional structure of the IclR family member AllR in complex with its effector glyoxylate was reported by Walker et al. (38). The alignment of AllR and TtgV sequences (24% identity) reveal that 4 of the 6 amino acids mutated in this study (Val-140, His-200, Val-204, Val-223) were found to correspond to amino acids (Leu-149, His-211, Leu-215, Ser-234, respectively) that are involved in glyoxylate binding by AllR. Furthermore, the mutation of each of His-211, Leu-215, and Ser-234 amino acids rendered AllR fully insensitive to 1 mM glyoxylate, as evidenced by EMSA. Based on the data reported for AllR and those obtained in this study for TtgV, we suggest that the amino acids mutated in this study in TtgV may be part of the effector-binding pocket of this repressor. Five of the six amino acids present in the effector-binding site of TtgV were hydrophobic. This contrasts with the results for AllR since of the 10 amino acids involved in glyoxylate binding, only two were hydrophobic. A similarly hydrophobic composition was found for other multi-drug-recognizing regulators such as QacR (23) and BmrR (19), which supports the notion that hydrophobic interaction is the main driving force in multidrug recognition.

Different Binding Modes for Monoaromatic and Biaromatic Effectors—The ITC studies of mutant proteins revealed altered affinity for effectors with respect to the wild-type protein. Indeed, TtgV variants bound monoaromatic compounds with a lower affinity than TtgV, whereas biaromatic compounds were recognized with a higher affinity (Table 1 and Fig. 3). In this respect, a clear parallelism exists with BmrR, a well studied transcriptional regulator with multidrug binding capacity (39). Based on the co-crystal structure of BmrR in complex with the effector tetraphenylphosphonium (40), Vázquez-Laslop et al. (19) prepared alanine mutants of amino acids involved in the binding of this effector and studied the interaction of the mutant proteins with six different effectors. With the exception of 3 key residues for which affinity decreased for all effectors, the remaining mutations caused an irregular pattern characterized by an increase in affinity for some effectors and a decrease for others. The authors concluded that each effector contacts with a different set of amino acids, giving rise to multiple effector binding modes. In this respect, TtgV appears to be different since a clear pattern emerged for the binding of mono- and bicyclic compounds. Our data are consistent with the existence of two different binding modes specific for either mono- or biaromatic compounds.

The central feature of the binding mode for monoaromatic compounds appears to be extensive contacts between the effector and Phe-134 and His-200 side chains. Besides the strong reduction in the binding constants of F134A and H200A for the monoaromatic compounds (Table 1), the respective enthalpy changes are much less favorable for both mutant proteins. This is exemplified by the enthalpy changes of 4NT binding to F134A and H200A, which, at –3.0 and –3.5 kcal/mol, were significantly below the value of the wild-type protein (–9.7 kcal/mol). A similar decrease in enthalpy is observed for BN binding to TtgVF134A (Table 1). Favorable enthalpy changes are typically attributed to the extent of direct contacts between the two ligands, and the decrease in enthalpy observed in alanine mutants has been used to estimate the contribution of single side chains to the binding energetics (41, 42). This observation is in consonance with previous reports that showed that phenylalanine side chains play essential roles in the recognition of monoaromatic compounds such as in toluene monooxygenase (43) or nitrotoluene dioxygenase (44).

The binding mode of bicyclic compounds is unlikely to involve close contacts between Phe-134 and His-200 and the effector. The replacement of both amino acids by alanine caused an increase in affinity, and no clear tendency was observed for the corresponding enthalpy changes (Table 1). Why then does the replacement of both of these residues, which are essential for the binding of the monoaromatic compounds, increase the affinity for the biaromatic ones? According to the model, the TtgV effector-binding site appears to be a closed, buried space with well defined lateral borders (Fig. 1A), forming a binding pocket that is 13 Å deep. Attempts to model 1NL in this pocket resulted in the conclusion that a degree of steric hindrance may exist that impedes the entrance or optimal accommodation of biaromatic compounds in the binding site. All mutations resulted in an increase in the volume of the binding pocket. This change, however, was particularly pronounced for F134A and H200A, causing increases from 1200 Å3 (wild type) to 1610 and 1540 Å3, respectively, as determined by PASS (28). This significant increase in volume is thought to relieve the steric constrains, enabling higher affinity binding due to the optimized accommodation of the effector. The volumes of the effector binding pocket of mutants F134V and H200V are in between the volumes of the corresponding alanine mutants and the wild-type protein. The fact that the affinities for bicyclic compounds for valine mutants also lie between the affinities observed for the alanine mutant and the wild-type protein supports the aforementioned hypothesis on the relief of steric constraints on volume increase. However, the analysis of mutant V140F demonstrates that the volume increase is not the sole determinant of the binding behavior of bicyclic compounds. 1-NL bound with significantly higher affinity to V140F than to V140A. This increase in affinity despite the decrease in the volume of the binding pocket is attributed to the establishment of additional van der Waals contacts between the hydrophobic side chain of Phe-140 and the effector. Van der Waals interactions were reported to play a key role in substrate of effector recognition in other multidrug-binding proteins (3, 5, 20, 21). In summary, data presented here reinforce the notion that multidrug-binding proteins have not evolved to recognize a given substrate with high affinity but to recognize instead a large range of structurally different compounds with physiologically relevant affinity.

Different Effectors Have Different Signal Transduction Efficiencies in Vitro, and Val-223 Might Play a Key Role—We were able to show, with EMSAs of wild-type TtgV in the presence of effector concentrations corresponding to either 1 or 10 x the KD, that the bicyclic compounds analyzed were more efficient than the monocyclic compounds in inducing release of the protein (Fig. 5A). This is consistent with the idea that the signal generated by the binding of biaromatic 1NL and IND is transmitted more efficiently than the signal transmitted by the two monoaromatic compounds. Furthermore, our data also suggest that the binding mode for biaromatic compounds may be associated with greater efficiency in triggering the release of the repressor.

The V223A mutation did not alter the affinity of the protein for operator DNA (Table 2). The experiments with ITC showed that TtgVV223A in its free and DNA-bound form binds 1NL with similar affinity (Fig. 4). We also performed EMSA studies of TtgVV223A in the presence of 1NL concentrations corresponding to 1–10 times the KD value (Fig. 5B). The data showed that TtgVV223A was less efficient in the release of the protein when compared with the wild-type protein (Fig. 5). We then hypothesized that the interdomain signaling cascade, in which the signal generated by the binding of an effector is transmitted to the DNA-binding domain, can be modulated by amino acid substitutions in the effector-binding site. This hypothesis is thus consistent with the notion that a single amino acid can have two roles that need not be necessarily related to each other, i.e. roles in effector binding and signal transmission. Mutation of such amino acids can have different effects with respect to both roles.

Indole as an Effector of Physiological Relevance—A central but often neglected question concerning multidrug-binding proteins is whether ligands are physiologically relevant. In this study, we have shown that the two-ring compounds 1NL and IND are substrates of the TtgDEF and TtgGHI efflux pumps (Table 3). Indole can be synthesized by P. putida and other microorganisms (45) and is excreted into the medium as a final metabolic product (46). Indole is toxic for other microorganisms, and incubation of E. coli with 5 mM IND led to a severe loss of viability (47). It was shown recently that IND induces the expression of multidrug exporter genes in E. coli, which confers acquired multidrug resistance (48). Some of the genes induced are highly homologous to pump genes controlled by TtgV, as exemplified by E. coli AcrD and AcrE, which share 53 and 44% sequence identity with TtgH and TtgG, respectively. Furthermore, it has been demonstrated that the E. coli AcrEF pump extrudes IND (49).

The toxicity of IND is particularly acute in dense microbial populations found in sewage, where concentrations in the millimolar range have been reported (50). In this context, IND can be considered an environmental pollutant (50, 51). P. putida DOT-T1E was isolated from sewage (1) in work aimed at isolating bacteria that exhibit tolerance to toluene. Subsequently, the TtgDEF and TtgGHI systems were shown to play a key role in toluene tolerance (2). It should be recalled that the mechanisms through which IND and toluene exert their toxicity are the same. They both dissolve in the membrane, leading to cell disorganization (47, 52). The parallels in the toxic mechanisms of these two aromatic compounds reflect their common chemical traits and might explain why TtgV, TtgGHI, and TtgDEF act on IND and toluene. The data we report here therefore suggest that one of the physiological reasons for the existence of the TtgV-TtgGHI/TtgDEF systems may be the extrusion of IND in dense bacterial populations, but whether the extrusion of other drugs (including toluene) occurs opportunistically is unknown.


   FOOTNOTES
 
* This study was supported by Grants GEN2001-4698-CO5-03, BIO2003-00515, and BIO2006-05668 from the Ministry of Science and Education in Spain, Grant VEM2004-08560 from the Ministry of the Environment, a grant from Junta de Andalucía (Andalusian Regional Government) to research group CIV191, and Grant RGY0021/2002 from the Human Frontier Program. 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. Back

1 To whom correspondence should be addressed: Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda, número 1, E-18008 Granada, Spain. Tel.: +34 958 181600, Ext. 326; Fax: +34 958 135740; E-mail: jlramos{at}eez.csic.es.

2 The abbreviations used are: 1NL, 1-naphthol; BN, benzonitrile; EMSA, electrophoretic mobility shift assay; IND, indole; ITC, isothermal titration calorimetry; MIC, minimal inhibitory concentration; 4NT, 4-nitrotoluene; PDB, Protein Data Bank. Back

3 W. Terán, A. Felipe, M.-E. Guazzaroni, T. Krell, R. Ruiz, J. L. Ramos, and M.-T. Gallegos, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We thank Pedro Mateo and Javier Ruiz from the University of Granada for help in recording the CD spectra, Ana Hurtado for DNA sequencing and competent cells, Sandy Fillet for purification of TtgV proteins, Carmen Lorente and Mai Fandila for secretarial assistance, and K. Shashok for improving the English in the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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A. Busch, J. Lacal, A. Martos, J. L. Ramos, and T. Krell
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