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J. Biol. Chem., Vol. 282, Issue 22, 16476-16491, June 1, 2007
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From the
Banting and Best Department of Medical Research, Toronto, Ontario M5G 1L6, Canada, Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L6, Canada, ¶Ontario Center for Structural Proteomics, Toronto, Ontario M5G 1L6, Canada, and ||Canada Structural Genomics Consortium, Toronto, Ontario M5G 1L6, Canada
Received for publication, November 22, 2006 , and in revised form, March 28, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-subunit (5). A third IclR-box was located between +14 and -21 bases of the iclR promoter (6). All three IclR-boxes identified in the promoters of aceB and iclR genes contain near-perfect palindromes. Although not identical, their sequences share a 15-mer consensus motif, 5'-TGGAAATNATTTCCA, which was identified by in vitro random oligonucleotide selection experiments (7). In growth media containing acetate, IclR is believed to release the DNA as a result of binding a small molecule. Although this molecule is likely related to a metabolite within the glyoxylate bypass pathway, the precise nature of this IclR effector remains nevertheless in doubt. Phosphoenolpyruvate was reported to alter IclR binding to DNA in vitro (8) but recently was shown to have no effect on IclR activity by Yamamoto and Ishihama (5).
IclR is the founding member of a large family (Pfam 01614, COG1414) of sequence-related microbial transcriptional regulators with more than 500 members identified in bacterial and archaeal genomes. Numerous bacterial genomes such as E. coli K12 (9) and Pseudomonas aeruginosa PA01 (10) contain multiple members of this family (8 and 9, respectively) with the largest number (40 members) identified so far in Bordetella bronchiseptica RB50 (11). The characterized members of this family are involved in the regulation of diverse catabolic pathways ranging from the degradation of plant cell polysaccharides in the plant pathogen Erwinia chrysanthemi (12) to the metabolism of aromatic acids in E. coli, Acinetobacter (13, 14), and Pseudomonas (15).
The characteristic structural features for members of this family, gained primarily from the crystal structure of TM0065 from Thermotoga maritime (PDB code 1MKM), are fully consistent with the role of transcription regulators responding to chemical stimuli (16). The TM0065 structure confirmed the presence of N-terminal classic-winged helix-turn-helix DNA binding domain as well as demonstrated that the effector binding C-terminal domain has significant structural homology to the PAS/GAF domain, a known small molecule binding motif (17, 18). The N- and C-terminal domains in the structure are connected by a linker helix, which together with N-terminal domain participates in protein dimerization. In the crystal lattice TM0065 dimers further oligomerize into tetramers, which appear to be the functional unit common for a number of IclR regulators (16). The C-terminal domain retains its structure and small molecule binding function when expressed without the DNA binding domain and was structurally characterized for three E. coli representatives of IclR family, KdgR (PDB code 1YSP), YiaJ (PDB code 1YSQ), and AllR3 (Ref. 20, PDB code 1TF1). Although sharing relatively low sequence identity between them and with previously characterized TM0065, all four structures were very similar, confirming a common structural framework for most of the IclR family members.
AllR is involved in transcriptional regulation of the allantoin catabolism (19) and shares 42% sequence identity with IclR. The AllR C-terminal domain structure was recently determined in the presence and absence of its chemical effector glyoxylate (20), providing the first detailed view of the molecular interactions of an IclR family representative with a small molecule. According to the AllR-glyoxylate complex structure, the location of the effector binding pocket in IclR regulators corresponded to the co-factor-accommodating cavity in PAS/GAF domains; however, the binding mechanisms were not conserved between these structurally similar domains (17, 18, 20).
Of the hundreds of IclR family members, only a few of the sensor chemicals have been identified, which apart from glyoxylate, include 3-(3-hydroxyphenyl) propionic acid for E. coli MhpR (21), 2-keto-2-deoxyglyconate for E. chrysanthemi KdgR (12), p-hydroxybenzoate and protocatechuate for Acinetobacter PobR and PcaU, respectively (13, 22), N-3-oxo-octanoyl homoserine lactone (3OC8HSL) for Agrobacterium tumefaciens AttJ (23), and 1-naphthol and 2,3-dihydroxynaphthalene for Pseudomonas putida TtgV (24). Thus, the effector specificity of hundreds IclR regulators, including the founding member of the family, remains uncertain.
To address these issues we used IclR as a test case and screened it against a large number of intermediates associated with glyoxylate bypass including phosphoenolpyruvate as part of a library of more than 150 metabolites by a high-throughput protein stability assay (25, 26). This methodology takes advantage of the well documented phenomenon of enhanced thermal stability of the protein in the presence of a specifically bound ligand (27). The molecules that had a stabilizing effect were further assessed for the effector role for IclR using biophysical and functional assays.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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For protein expression and purification the iclR gene was amplified from wild type E. coli BW25113 chromosomal DNA by PCR. To identify the boundaries of the ligand binding domain of IclR, a multiple sequence alignment of IclR and its homologues was performed. The link between the DNA and ligand binding domains in E. coli IclR was predicted to lie between amino acids Ser 98 and Leu 101. Two fragments corresponding to the 176 and 173 C-terminal amino acids were amplified and tested for expression of the soluble domain as previously described (20). The expression of a fragment of iclR corresponding to amino acids 98–274 yielded a soluble domain of IclR (C-IclR) that was used for further experiments. All amplified fragments were cloned into the NdeI and BamHI sites of a modified form of pET15b (EMD Biosciences) in which a TEV protease cleavage site replaced the thrombin cleavage site and a double stop codon was introduced downstream from the BamHI site. This construct provides for an N-terminal hexahistidine tag separated from the protein by a tobacco etch virus protease recognition site (ENLYFQ
GS).
Construction of Deletion Mutants—An IclR deletion mutant was generated by the methods described by Datsenko and Wanner (29). To prepare competent cells for transformation, BW25113 (lacIq rrnBT14 
lacZWJ16 hsdR514 araBADAH33 rhaBADLD78; 29) containing pKD46 was cultured at 30 °C in SOB broth (28) containing 100 µg of ampicillin/ml. When the A600 reached 0.5, the culture was centrifuged at 4000 rpm for 5 min, and the cells were washed 3 times with cold water before being resuspended in 1% of the original culture volume of water. PCR methods were used to amplify the kanamycin resistance gene (km) from pKD4 by using the primers iclRFw (5'-GTTCAACATTAACTCATCGGATCAGTTCAGTAACTATTGCATTAGCTAACGTGTAGGCTGGAGCTGCTTC-3') and iclRrv (5'-GCGATTAACAGACACCCTTATTCTATTGCCACTCAGGTATGATCATATGAATATCCTCCTTA-3'). The PCR products were purified with a Qiagen kit, treated with DpnI, and repurified by electrophoresis. The km gene was transformed into BW25113-competent cells by electroporation (Gene Pulser; pulse controller at 200 ohms, capacitance at 250 microfarads, and voltage at 25 kV). After electroporation, the cells were grown with shaking in 1 ml of SOC (28) medium at 37 °C for 1 h, and the cultures were plated onto Luria-Bertani (LB) agar containing 25 µg of kanamycin/ml. The Kmr transformants were purified on new kanamycin-LB plates. The mutants in which the target genes were replaced by the km gene were verified by PCR using the primers iclRcfw (5'-CTCATCGGATCAGTTCAGTAAC-3') and iclRcrv (5'-CTTATTCTATTGCCACTCAGG-3'). To delete the km gene from the chromosome, pKD46 was removed from the cells by growing the bacteria at 37 °C, and then pCP20, expressing the Flip recombinase, was introduced by transformation. The transformants containing pCP20 were grown overnight with shaking at 42 °C, and the cultures were plated on LB agar without antibiotics. Colonies were tested for sensitivity to kanamycin and ampicillin. crp mutants were constructed by P1 transduction (30) using the strain JW3320 as donor (National BioResource Project, National Institute of Genetics, Mishima, Japan (31)).
Real-time Quantitative PCR Studies—Bacterial cells were cultured in MOPS minimal media (32) with 10 mM pyruvate as carbon source, and when required, 10 mM glyoxylate was added. When the A600 reached 0.5, cells were collected by centrifugation at 4 °C. Total RNA was subsequently isolated with RiboPureTM-Bacteria (Ambion) in accordance with the manufacturer's protocol. cDNAs were synthesized with the superscript first-strand synthesis kit (Invitrogen) in accordance with the manufacturer's instructions and stored at -80 °C before use. Real-time quantitative PCR was carried out on the Applied Biosystems 7300 apparatus (Applied Biosystems) using Platinum® SYBR® Green qPCR SuperMix UDG (Invitrogen) in accordance with the manufacturer's recommended protocol. Primers used for the real-time PCR were as follows: for rrsC, 5'-CAGCCACACTGGAACTGAGA-3' and 5'-GTTAGCCGGTGCTTCTTCTG-3'; for aceB, 5'-CTGCGTGACCATATTGTTGG-3' and 5'-CAGGCGTGAGTAAGCATTCA-3'; for aceK, 5'-TTCGTGCCTGCTATCAACTG-3' and 5'-AGATATTGAGCGGCACCATC-3'.
Protein Purification—The His-tagged fusion proteins were overexpressed in E. coli BL21-Star(DE3) cells (Stratagene) harboring an extra plasmid encoding three rare tRNAs (AGG and AGA for Arg, ATA for Ile). The cells were grown in LB at 37 °C to an A600 
0.6, and expression was induced with 0.4 mM isopropyl 1-thio-
-D-galactopyranoside. After the addition of isopropyl 1-thio--D-galactopyranoside, the cells were incubated with shaking at 15 °C overnight. The cells were harvested, resuspended in binding buffer (500 mM NaCl, 5% glycerol, 50 mM HEPES, pH 7.5, 5 mM imidazole), flash-frozen in liquid N2, and stored at -70 °C. The thawed cells were lysed by sonication after the addition of 0.5% Nonidet P-40 and 1 mM each of PMSF and benzamidine. The lysate was clarified by centrifugation (30 min at 17,000 rpm) and passed through a DE52 column preequilibrated in binding buffer. The flow-through fraction was then applied to a metal chelate affinity column charged with Ni2+. After the column was washed, the protein was eluted from the column in elution buffer (binding buffer with 500 mM imidazole). The hexahistidine tag was then cleaved from the protein by treatment with recombinant His-tagged tobacco etch virus protease. The cleaved protein was then resolved from the cleaved His tag and the His-tagged protease by flowing the mixture through a second Ni2+ column.
The purified proteins were dialyzed against 10 mM HEPES, pH 7.5, 500 mM NaCl and concentrated using a BioMax concentrator (Millipore). Before crystallization, any particulate matter was removed from the sample by passage through a 0.2-µm Ultrafree-MC centrifugal filtration device (Millipore). Selenomethionine-labeled proteins were expressed using the same vector and host strain but in supplemented M9 media (33). The sample was prepared under the same conditions as the native protein except for the addition of 5 mM -mercapto-ethanol to the purification buffers.
Size-exclusion Chromatography—100 µl of protein sample contained 10 mM HEPES, pH 7.5, 500 mM NaCl, 25 µM IclR wild type or its mutant derivatives, and when indicated, 1 mM glyoxylate or 10 mM pyruvate. After 20 min of incubation on ice, samples were injected onto a Superose 12 10/300 GL gel filtration column (Amersham Biosciences) installed on anÁkta system (Amersham Biosciences) equilibrated with 10 mM HEPES, pH 7.5, 500 mM NaCl. Filtration was performed at 4 °C at a flow rate of 0.5 ml/min, and the protein concentration was monitored by measuring the absorbance at 280 nm. Blue dextran 2000 was used to determine the void volume. A mixture of protein molecular mass standards containing-amylase (200 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa) was applied to the column under similar conditions. The elution volumes and molecular masses of the protein standards were used to generate a standard curve from which the apparent molecular mass was determined.
Crystallization and Data Collection—The primary crystallization condition was determined by a sparse crystallization matrix (Hampton research kits Crystal Screen 1 and PEG/Ion Screen) at room temperature using the sitting drop vapor diffusion technique in 96-well plates. This condition was optimized by varying the pH and the concentration of the solutes. For apoIclR the best crystals were obtained using hanging drops in 0.2 M potassium acetate, pH 7.0, 20% polyethylene glycol 3350 for 2–5 days at room temperature. For diffraction studies, the crystals were flash-frozen with the crystallization buffer plus 30% ethylene glycol as cryoprotectant. For the complex structures the crystals obtained in the crystallization condition described above were soaked in 10 mM solution of either glyoxylate or pyruvate for 1 h. For diffraction studies, the crystals were flash-frozen with the crystallization buffer plus 25% ethylene glycol as the cryoprotectant.
Diffraction data of crystals of C-IclR were collected at 100 K at the 19ID beamline of the Structural Biology Center at the Advanced Photon Source, Argonne National Laboratory. The absorption peak and rising inflection point were determined by calculating and plotting f' and f'' values against energy (34) from the fluorescence spectrum. The three-wavelength anomalous diffraction data were collected from a Se-Met-substituted protein crystal using an inverse beam strategy. All crystallographic data were measured with the custom-built 3 x 3-tiled CCD (charge-coupled device) detector (35). The experiment, data collection, visualization, and processing were controlled with the HKL2000 program package (36). Statistics of data collection and processing are provided in Table 3. Diffraction data for glyoxylate and pyruvate complexes were collected at home source (Rigaku FR-E generator with Raxis4++ detector), and the data were integrated and scaled with *TREK (37).
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Electrophoretic Mobility Shift Assays (EMSA)—Electrophoretic mobility shift assays for IclR and mutant proteins were performed using proteins purified and concentrated according the procedures described above. A fragment of the aceBAK promoter region, from position -117 to +17 (containing IclR box II), was generated by PCR using biotin-prelabeled (5'-end) primers and purified using QIAquick spin columns (Qiagen). Incubation mixtures for EMSA (20 µl) contained 2.5 nM 5'-labeled DNA fragment, 50 mM Tris-HCl, pH 7.5, 150 mM KCl, 10 mM MgCl2, 0.01% Triton X-100, 50 ng/µl poly(dI-dC)-nonspecific competitor DNA, purified IclR protein (0–100 nM), and ligand (0–1 mM) where indicated.
After incubation for 20 min at 37 °C, samples were separated on 5% acrylamide-bisacrylamide nondenaturing gels in 0.5x Tris borate-EDTA buffer, pH 8.3 (TBE). Electrophoresis was performed at 100 V using ice-cold 0.5x TBE as a running buffer, and DNA was transferred from the polyacrylamide gel to the Biodyne B Positive Nylon Membrane (Pierce) by electroblotting at 380 mA for 40 min in 0.5x TBE. Transferred DNA was cross-linked for 15 min using UV cross-linker equipped with 312-nm bulbs. Biotin-labeled DNA was detected by horseradish peroxidase/Super Signal detection system (Pierce). Membranes were exposed to Eastman Kodak Co. x-ray film. For quantitative EMSA, the distribution of bound and unbound DNA was quantified using ImageJ (National Institutes of Health).
In Vitro Transcription Runoff Assays—For in vitro transcription runoff the complete aceB promoter was amplified from position -297 to +434 and cloned in a derivative of the pGF-Puv vector (Clontech) in which the gfp gene was replaced with the aceB promotor in the SapI-EcoRI position. The expected specific transcript was 434 bases long. The recombinant plasmids were amplified in E. coli DH5, cut with EcoRI, and gel-purified. The reaction mixtures (20 µl) contained template DNA (5 nM), purified IclR or its mutant forms (100 nM), and glyoxylate (1–1000 µM) in a buffer consisting of 40 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM MgCl2, 0.01% Triton X-100, and 1 unit of RNase inhibitor. The reaction mix was preincubated for 10 min at 37 °C, and 20 nM RNA polymerase (Epicenter) was added. The reaction mix was further incubated for 5 min at 37 °C, and transcription was initiated by adding 2 µl of a mixture containing 2 mM each ATP, GTP, and CTP, 1.5 mM UTP, and 0.5 mM biotin-11-UTP (PerkinElmer Life Sciences). After 20 min at 37 °C, the reactions were terminated with 10 mM EDTA. The ethanol-precipitated transcripts were analyzed on 6% acrylamide, 7 M urea gels. Electrophoresis was performed at 100 V using ice-cold 0.5x TBE as a running buffer, and RNA was transferred from the polyacrylamide gel to the Biodyne B positive nylon membrane (Pierce) by electroblotting at 380 mA for 40 min in 0.5x TBE. Transferred RNA was cross-linked for 15 min using UV cross-linker equipped with 312-nm bulbs. Detection was performed as described above. Preheated biotinylated 2-log DNA ladder (BioLabs) was used as molecular weight marker.
Screening Using Static Light Scattering—Purified C-IclR protein was screened against a library of 160 compounds using static light scattering (StarGazer) (45). Protein samples were diluted to a final concentration of 0.4 mg/ml in 100 mM Hepes, pH 7, 150 mM NaCl (26). 50-µl aliquots of protein solution containing the chemical compounds at 1 mM were placed in duplicate into clear-bottom 384-well plates (NuncTM, Nalgene Nunc International) and heated from 27 to 80 °C at the rate of 1 °C/min. The formation of protein aggregates was monitored by static light scattering. Images of scattered light were captured every 30 s, and the light intensities were translated to arbitrary numbers using StarGazerTM proprietary software. Intensities were plotted against temperature for each sample well, and transition curves were fitted using the Boltzmann equation. The midpoint of each transition was calculated and compared with the one calculated for the reference sample. If the difference between them was greater than 1.5 °C, the corresponding compound was considered to be a "hit," and the experiment was repeated to confirm the effect.
Circular Dichroism (CD) Spectroscopy—CD spectra were collected at 25 °C on a AVIV 62D CD spectrophotometer from 200 to 260 nm using a 0.1-mm path length cell with a scan rate of 100 nm/min, a time constant of 1.0 s, a bandwidth of 1 nm, and a sensitivity of 100 millidegrees. Each spectrum was the average of 10 scans. After background subtraction, the spectrum was expressed in molar ellipticity. The proteins were analyzed at a final concentration of 1 mg/ml, and the assays were performed in 10 mM HEPES, pH 7.5, 0.5 M NaCl. When necessary, glyoxylate or pyruvate was added at 1 mM.
Isothermal Titration Calorimetry—Measurements were performed on a VP-Microcalorimeter (MicroCal, Northampton, MA) at 25 °C. The protein was thoroughly dialyzed against 10 mM HEPES, pH 7.5, and 500 mM NaCl. Solutions of glyoxylate (0.5 mM) and pyruvate (1.5 mM) were directly prepared in dialysis buffer. Each titration involved a series of 4-µl injections of effector molecules into the protein solution. 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 (46). From the curve thus fitted, the parameters H (reaction enthalpy), KA (binding constant, KA = 1/KD), and n (reaction stoichiometry), were determined. From the values of KA and H, the changes in free energy (G) and in entropy (S) were calculated with the equation G = -RT ln KA =H - TS, where R is the universal molar gas constant, and T is the absolute temperature.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-ketoglutarate, succinate, and fumarate as well as phosphoenolpyruvate. The glyoxylate binding domain of AllR regulator, C-AllR (20), was also tested against the same library as a control. The thermal melt conditions for C-IclR and C-AllR were established to generate interpretable unfolding data (described under "Experimental Procedures"). The compounds that induced a shift in the midpoint transition temperature (Tm) of proteins by more than 1.5 °C are listed in Table 1.
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Tm in the presence of glyoxylate (11.1 °C) was close to the effect of this compound (9.9 °C) on C-AllR. Additional compounds inducing significant stabilization of C-IclR included allantoate, pyruvate, and phosphoenolpyruvate. Apart from glyoxylate, allantoate, DL-glyceraldehyde 3-phosphate, and phosphoenolpyruvate, C-AllR was stabilized by allantoin but not by pyruvate (Table 1). The thermal stabilizing effect of glyoxylate on C-IclR was confirmed by circular dichroism spectroscopy (Fig. 1). In agreement with the differential light-scattering results, the circular dichroism analysis showed an increase in 12 °C in the melting temperature of C-IclR in the presence of glyoxylate. All five compounds identified for C-IclR contained a common chemical motif in the form of adjacent carbonyl or hydroxyl groups (Table 1) with the strongest thermal stabilizing agent, glyoxylate, being the simplest representation of this chemical scaffold. The intermediate role played by glyoxylate in the pathway regulated by IclR also strengthens its case as a candidate for the native IclR effector molecule. At the same time identification of several metabolites causing IclR thermal stabilization and having a common chemical signature indicates that this sensor protein might be reactive to more than one effector molecule.
Effect of Small Molecule Modulation of IclR Binding in Vitro— The effect of glyoxylate, allantoate, DL-glyceraldehyde-3-phosphate, pyruvate, and phosphoenolpyruvate on IclR activity was tested in vitro by EMSA. The binding of IclR to the DNA fragment corresponding to the promoter region of the aceBAK operon and containing IclR box II (5) was analyzed in the presence and the absence of 100 µM or 1 mM concentrations of each compound. According to EMSA results, glyoxylate caused dissociation of the IclR regulator from DNA, thus increasing the fraction of unbound DNA (Fig. 2). On the contrary, pyruvate and, to a lesser extent, DL-glyceraldehyde 3-phosphate and phosphoenolpyruvate stabilized the IclR-DNA complex and resulted in a decrease of unbound DNA (Fig. 2). Allantoate did not affect the IclR/aceB complex (data not shown).
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subunit, as described earlier (5). Phosphoenolpyruvate and DL-glyceraldehyde 3-phosphate were able to stabilize the IclR-DNA complex (Fig. 2); however, the concentrations required to obtain this effect (over 10 mM, data not shown) were out of the physiological range for these compounds (47–50). Thus, phosphoenolpyruvate and DL-glyceraldehyde 3-phosphate were not further considered for an IclR effector role.
Because glyoxylate and pyruvate had distinctive and antagonist effects on the formation of the IclR-DNA complex, in the next series of EMSA experiments we analyzed the combined effect of glyoxylate and pyruvate on the stability of the IclR-DNA complex. In this setting one of compounds was added in increasing amounts, whereas the concentration of the other was kept constant. The results obtained (Fig. 4A) demonstrated that the glyoxylate-induced dissociation of the IclR-operator complex was overturned by a 50-fold excess of pyruvate (500 µM pyruvate with 10 µM glyoxylate). These results were corroborated by doing the reciprocal experiment (Fig. 4B). The common chemical features of these compounds suggested that this effect might be due to the competition for the same binding site in IclR, with glyoxylate having a significantly higher binding affinity than pyruvate.
To further characterize IclR interactions with potential effector molecules, the thermodynamic properties of IclR interactions with glyoxylate and pyruvate were determined using isothermal titration calorimetry. The titration of C-IclR with each compound followed an exothermal heat change profile, giving rise to a sigmoidal binding curve with glyoxylate or hyperbolic with pyruvate (Fig. 5). The calculated thermodynamic parameters are summarized in Table 2. The stoichiometry of the reaction, 0.5, is consistent with the binding of one ligand molecule per IclR dimer. In accordance with EMSA results, the C-IclR dissociation constant (KD) for glyoxylate (0.9 ± 0.2 µM) was significantly lower (150 times) than that for pyruvate (156.2 ± 7.9 µM). Although the KD values obtained for binding of glyoxylate and pyruvate to the full-length IclR were higher (14.2 ± 1.2 and 321.7 ± 20 µM, respectivelytively), their relative ratio was within the same range as obtained for C-IclR.
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Modulation of IclR Binding by Glyoxylate in Vivo—To assess the effect of glyoxylate on IclR activity in vivo, the expression level of aceBAK operon was measured during growth on minimal media in the presence and absence of this compound. Although early genetic studies identified IclR as the main regulator of aceBAK operon (51), recent E. coli transcriptome analysis indicated that this operon is also under control of the global catabolite regulation protein, Crp. The Crp potential binding sequence was found overlapping the IclR box II (52). In light of this new data, the IclR activity was monitored in the presence and the absence of a functional Crp regulator.
Single iclR and crp as well as double (iclRcrp) deletion strains were prepared in the E. coli BW25113 strain background. Deletion strains were grown to exponential phase on MOPS minimal medium with pyruvate (10 mM) as the carbon source in the presence and in the absence of 5 mM glyoxylate. The expression levels of aceB and aceK genes were measured using real-time quantitative PCR. The expression profiles of both genes followed the same trend; thus, only the results for aceB gene will be discussed further on.
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iclR and crp strains was increased by 3.3- and 24-fold, respectively, compared with the wild type strain. The double iclRcrp deletion caused a 31-fold increase in aceB expression corresponding to the combined effect of each gene deletion. The presence of glyoxylate in the media had no significant effect on the level of aceB expression in iclR and iclRcrp strains lacking the functional IclR regulator, whereas it triggered a 1.4-fold increase when the IclR regulator was present in the cells (crp strain) (Fig. 6). The glyoxylate-mediated induction was dose-dependent since in the presence of 10 mM glyoxylate a 2.7-fold induction in aceB expression was observed (data not shown). In the case of the wild type strain containing both IclR and Crp regulators, the presence of glyoxylate in the media did not result in a detectable increase of aceBAK, expression probably due to effector-independent tight repression by the functional Crp regulator.
The substantial derepression effect of the Crp deletion confirmed the major role played by this global regulator on the expression of the aceBAK operon. The effect of the IclR deletion was significantly smaller yet consistent with its role as a specific regulator of this operon. The derepression effect in the presence of glyoxylate observed in the crp strain appears to be specific to the presence of functional IclR, indicating that glyoxylate can affect the activity of the IclR regulator in a manner expected from the native effector molecule. Unfortunately we were unable to study the corepressor effect of pyruvate in vivo since the crp deletion strain is unable to grow on the variety of carbon sources (52) required for these kinds of studies.
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sheets and -helices are given in relation to the full-length homolog TM0065 (16).
The C-IclR domain displays a similar architecture to the two other IclR family members whose structures have been determined, TM0065 (16) and AllR (20). The C-IclR structure comprises a centrally located six-stranded anti-parallel -sheet surrounded on one side by two long -helices (5 and 9) and on the other by three shorter -helices (6–8). The -sheet is strongly curved, with the shortest helix (6) fitting on the inside of the half-barrel (Fig. 7).
In complex structures both the glyoxylate and pyruvate molecules occupied the cavity corresponding to the effector binding pocket in the AllR structure (20). The map quality was assessed by constructing an omit Fo - Fc electron density map. The map was calculated after 10 cycles of refinement without the ligand in the model (Fig. 8). Similar to the C-AllR-glyoxylate complex structure, no significant conformational changes were observed in C-IclR complexes with pyruvate or glyoxylate when compared with C-IclR apo structure. The ligands were bound in the same orientation with almost identical positions for atoms C1, C2, O1, O2, and O3 (Fig. 8). Oxygen atoms of glyoxylate and pyruvate were coordinated by an extensive network of hydrogen bonds with the side chains and backbone of the protein. For each ligand the O1 atom is hydrogen-bound to the Asp-212O
2 and Gly-160N, the O2 atom to the Ser-239O
and Ser-241O, and the O3 to the Ala-161N. The C1-Sg distance varies from 1.86 to 2.09 Å for glyoxylate complex and from 1.90 to 1.97 Å for the pyruvate complex. Both complex models were refined with the same restraints with no covalent bond between ligand C1 and Cys-222S in the dictionary. In agreement with previous sequence analysis, all but two amino acids, Ile-134 and Leu-143, in the effector binding area are conserved between IclR and AllR (20). The corresponding positions in AllR are represented by leucine (Leu-129) and methionine (Met-138), respectively (Fig. 9).
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1 to C2, 3.68 versus 4.44 Å; C2 to C1, 4.17 versus 6.16 Å). In addition, the average distances between protein atoms are shorter by an average of 1 Å in C-IclR-glyoxylate versus C-IclR structures (10.8 and 11.6 Å, respectively), whereas there is no such difference between C-AllR and C-AllR-glyoxylate structures (11.96 and 12.1 Å, respectively) for the same parameters. These structural dissimilarities between binding cavities in C-IclR and C-AllR may contribute toward close to a 10-fold difference in KD for glyoxylate as determined by isothermal titration calorimetry (Table 2).
Taking into account the almost identical position of glyoxylate and pyruvate in the C-IclR structures, the interactions involving the methyl group of pyruvate, which is absent in glyoxylate, become critical in understanding the different effects of these compounds on IclR activity. The methyl group is well defined in the pyruvate electron density map (Fig. 8B) and is oriented toward the side chain of the Leu-143 residue belonging to the other molecule of the C-IclR dimer. The distances between C3 of the pyruvate and C2 atom of Leu-143 are on average 4.24 ± 0.4 Å for the four molecules in the C-IclR asymmetric unit. Leu-143 makes part of the hydrophobic patch formed by side chains of Met-146, Leu-154, His-216, and Leu-220, which are in the range of van de Waals interactions with the pyruvate methyl group. The presence of methionine instead of leucine in the corresponding position in AllR would explain why pyruvate had no stabilization effect (Table 1).
In C-IclR the interdomain interactions between C-IclR monomers are similar to those observed in the C-AllR and TM0065 structures. The C-IclR and C-AllR dimers are superimposable to a root mean square deviation (r.m.s.d.) of 1.75 Å, compared with an r.m.s.d. of 1.01 Å for individual subunits. This common arrangement of the C-terminal domains of three different IclR family members in three different unit cells supports the physiological relevance of these interactions for tetramerization of IclR regulators (Fig. 7B). The additional intermolecular interactions between the pyruvate methyl group and hydrophobic patch observed in the C-IclR-pyruvate complex structure would, thus, stabilize the tetramerization state of IclR protein.
Effect of Glyoxylate and Pyruvate on the Oligomerization of IclR—To establish the effect of glyoxylate or pyruvate on the oligomeric state of IclR protein, this protein was preincubated with 1 mM glyoxylate or 10 mM pyruvate and analyzed by gel filtration. The untreated IclR protein was used as the control.
In the absence of effector molecules IclR was eluted in two peaks corresponding to the dimeric (60 ± 5 kDa) or tetrameric (120 ± 12 kDa) states. The abundance of both fractions (54/46% relative percentage, respectively) indicated that under our experimental conditions the IclR protein is in a dynamic equilibrium between these two oligomeric states (Fig. 10, Table 4). After preincubation with glyoxylate, IclR was eluted predominantly in the form of the lower (60 kDa) molecular mass fraction, indicating a dramatic shift of equilibrium toward the dimeric state. On the other hand, preincubation with 10 mM pyruvate resulted in elution of IclR in the form of the higher (120 kDa) molecular mass fraction corresponding to the tetramer. Similar results were obtained if glyoxylate was included in the elution buffer; however, the tetrameric state of IclR was more favorable if pyruvate was included in the elution buffer. These results can be explained by the difference in affinity between the ligands (Table 2). Overall, size exclusion experiments demonstrated that glyoxylate and pyruvate have a dramatic effect on the oligomeric state of the IclR regulator. This suggests that the glyoxylate effect on IclR activity is due to the destabilization of tetramers, which is required for efficient binding to the operator DNA, whereas the co-repressor effect observed for pyruvate might be due to the stabilization of the IclR tetramer on the DNA.
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The alanine substitution of Leu-143 did not significantly affect the gel filtration profile of IclR (Fig. 11). However, according to the EMSA experiments a higher concentration (100 nM) of the L143A mutant protein was required to achieve the DNA binding comparable with the wild type IclR (Table 4). The addition of pyruvate stabilized complex of this mutant with operator DNA similarly to the wild type IclR, whereas the glyoxylate had a significantly lower effect on the L143A mutant (Fig. 12). In the presence of the latter ligand an additional band was observed on the EMSA gels. This additional band migrated faster than the one corresponding to the wild type IclR-DNA complex but slower that the free DNA (Fig. 12). This band would correspond to an IclR-DNA complex with a lower molecular weight than in case of the wild type IclR. Taking into account that IclR might bind DNA as a tetramer (5), this lower molecular weight complex would correspond to the association of operator DNA with the dimers of L143A as an intermediate in the releasing of IclR from the protein-DNA complex.
The gel filtration profiles of the alanine substitutions of Met-146, Leu-154, and Leu-220 showed them predominantly as dimers in solution (70/30 relative percentage, respectively, Table 4); however, they were still able to tetramerize over DNA as evidenced by a high molecular weight migrating complex (Fig. 12). The mutant proteins showed a significant variation of responses to glyoxylate and pyruvate (Table 4). Although the M146A mutant response to these ligands was similar to the wild type IclR protein, the L220A mutant showed no response to glyoxylate but was stabilized by pyruvate. The L154A mutant, to the contrary, responded to glyoxylate but not to pyruvate. These results put in evidence the different involvement of these amino acids in ligand binding.
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EMSA experiments on M146D showed low and high molecular weight protein-DNA complexes (Fig. 12). In agreement with the mutations to alanine, M146D does not interfere in the binding of glyoxylate or pyruvate. Moreover, glyoxylate disrupted the formation of the high molecular weight protein-DNA complex, increasing the amount of the lower molecular weight complex (Fig. 12). The presence of pyruvate, on the contrary, increases the formation of the high molecular weight complex. These results show that although the Met-146 is important for the stabilization of tetramers in solution, it is not directly involved in the binding of the ligands, although it might contribute to the overall hydrophobicity of the area.
In the case of L154D, the EMSA profile was similar to that of the wild type IclR; however, the response to glyoxylate was weaker (Fig. 12). Pyruvate had no stabilization effect on this mutant protein (Fig. 12), confirming the Leu-154 role in the specific interactions with pyruvate.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Taking into account the physiological data, we could hypothesize that the cell could maximize the efficiency of expression/repression of the aceBAK operon by using two antagonistic effectors on the same transcription factor. By EMSA and biophysical tests we were able to establish that IclR binds glyoxylate and pyruvate. Pyruvate is the first non-phosphorylated intermediate in glycolysis and a key intermediate in catabolic and biosynthetic reaction pathways. There are several pathways that deliver this compound (48, 72, 73). Siddiquee et al. (48, 49) have shown that the intracellular concentration of pyruvate can be from 0.15 to 0.4 mM depending if samples were taken from batch or continuous cultures. These values are within the affinity constants defined for IclR binding. Glyoxylate, on the other hand, is an important intermediate of the central microbial metabolism in the glyoxylate bypass. Glyoxylate is also generated from glycolate or purine degradation (74, 75). The intracellular concentration for this metabolite in E. coli has not been reported; however, there are studies in the closely related bacterium Salmonella typhimurium (76) which suggest that the concentration of glyoxylate should be very low (10 µM). Thus, the big difference in affinity between glyoxylate and pyruvate binding to IclR mig
Fo - Fc omit map was built using the REFMAC5 program (
