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Rational Tuning of Superoxide Sensitivity in SoxR, the [2Fe-2S] Transcription Factor; Implication of Species-Specific Lysine Residues Mayu Fujikawa, Kazuo Kobayashi, Yuko Tsutsui, Takahiro Tanaka, and Takahiro Kozawa Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01096 • Publication Date (Web): 19 Dec 2016 Downloaded from http://pubs.acs.org on December 26, 2016
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Rational Tuning of Superoxide Sensitivity in SoxR, the [2Fe-2S] Transcription Factor; Implication of Species-Specific Lysine Residues Mayu Fujikawa, Kazuo Kobayashi*, Yuko Tsutsui, Takahiro Tanaka, and Takahiro Kozawa The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki Osaka 567-0047, Japan Corresponding Author *E-mail:
[email protected] Telephone : +81-6-6879-8501
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ABBREVIATIONS SOD, superoxide dismutase; SOR, superoxide reductase; SoxRred, reduced ([2Fe-2S]+) SoxR; SoxRox, oxidized ([2Fe-2S]2+) SoxR; IPTG, isopropyl-β-D-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PQ, paraquat;
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ABSTRACT: In Escherichia coli, the [2Fe-2S] transcriptional factor, SoxR, functions as a sensor of oxidative stress. The transcriptional activity in SoxR is regulated by the reversible oxidation and reduction of [2Fe-2S] clusters. We previously proposed that superoxide (O2·-) has a direct role as a signal for E. coli SoxR, and that the sensitivity of the E. coli SoxR response to O2·- is 10-fold higher than that of Pseudomonas aeruginosa SoxR. The difference between the two homologues reflects inter-species differences in the regulatory role of O2·- activation. In order to investigate the determinants of SoxR’s sensitivity to O2·-, we substituted several amino acids that are not conserved among enteric bacteria SoxR homologues, and investigated SoxR interaction with O2·- using pulse radiolysis. The substitution of E. coli SoxR Lys residues 89 and 92 with Ala residues (K89AK92A), located close to [2Fe-2S] clusters, dramatically affected this protein’s reaction with O2·-. The second-order rate constant of reaction was 3.3 x 107 M-1 s-1, which was 10 times smaller than that of wild type SoxR. Conversely, the corresponding substitution of Ala90 with Lys in P. aeruginosa SoxR approximately 10-fold increased the rate. In contrast, introductions of the substitution Arg127Ser128Asp129→ Leu127Gln128Ala129 to E. coli SoxR, and the corresponding substitution (Leu125Gln126Ala127→Arg125Ser126Asp127) to P. aeruginosa SoxR, did not affect the reaction rates. In addition, the Lys mutation in E. coli SoxR (K89AK92A) showed a defect in vivo transcriptional activity by measuring βgalactosidase expression in response to paraquat. Our findings clearly support Lys is critical to response to O2·- and further transcriptional activity of SoxR.
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Bacteria possess molecular biosensors to respond to stressful environmental changes with exquisitely coordinated gene regulation.1-6 In Escherichia coli and related bacteria, the transcription factor SoxR mediates an oxidative stress response to superoxide and redox-cycling compounds. This unique protein contains a [2Fe-2S] cluster essential for its transcriptionenhancing activity, which is caused by undergoing structural changes between the oxidized and reduced forms.7-11 The soxR gene is conserved across diverse species of bacteria, both Gram positive and Gram negative.12 Sequence comparison of SoxR proteins reveals a highly conserved DNA binding domain and SoxR-specific cysteine motif CI[G/Q]CGC[L/M][S/L]XXXC, required for binding of the [2Fe-2S] cluster (Figure 1-A). Despite their sequence similarity, SoxR homologs have been shown to play distinct roles in different organisms. Unlike in E. coli and related enteric bacteria, the soxR regulons in Psudomonas aerginosa and Streptomyces coelicolor lack a gene typically involved in superoxide (O2·-) resistance and detoxification.12-15 Many of these bacteria produce redox-active pigments such as pyocyanin12, 15 and actinorhodin.16, 17 These SoxR target genes encode transporters, oxygenases, dehydrogenases, putative acetyl- or metyltransferases, all of which are potentially involved in the transformation or transport of small molecules, such as antibiotics.12, 15, 18 We previously reported to show the direct reaction of O2·- with the reduced form of SoxR (SoxRred) by the use of pulse radiolysis.19
This reaction (1) yields the oxidized form of SoxR (SoxRox). (As written in reaction (1), [2Fe2S]2+ and [2Fe-2S]1+ are oxidized and reduced forms of SoxR, respectively).19 Remarkably, the sensitivity of the E. coli SoxR response to O2·- (5 ×108 M-1 s-1) is 10-fold higher than that of P. aeruginosa response (4 ×107 M-1 s-1), despite the homology of E. coli SoxR to P. aeruginosa SoxR (62 % sequence identity; 77 % sequence similarity).12 The difference between the two
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responses reflects distinct regulatory roles for the physiological activation of O2·-. A crucial unanswered question concerns the mechanism underlying SoxR’s different sensitivities to O2·-. It is important to explore the O2·- sensing mechanisms of these proteins. X-ray crystallographic structures of oxidized E. coli SoxR have been determined at 2.8 and 3.2 Å resolutions, bound to the soxS promoter and free of the promoter, respectively (Figure 1-B).20 The SoxR protein consists of a DNA binding domain, a dimerization helix, and an Fe-S cluster domain. The [2Fe-2S] cluster of SoxR is coordinated by conserved four residues (Cys-119, Cys122, Cys-124, and Cys-130). The cluster is nearly completely exposed to the solvent. It is likely that this characteristic of this [2Fe-2S] cluster allows SoxR’s response to O2·-,19 multiple redox compounds,21, 22 and NO.23 Our UV resonance Raman studies showed that the redox state of the [2Fe-2S] cluster in SoxR is transmitted to the DNA-binding domain, resulting in a large conformational change of the target promoter.24 Comparison of the SoxR sequences from enteric and non-enteric bacteria reveals that there are several hypervariable sequences in the vicinity of the [2Fe-2S] cluster (Figure 1-A). It is interesting that SoxR homologues from enteric species contain two Lys residues, which are replaced by Ala residues in SoxR of almost all non-enteric species (Figure 1-A). The crystal structure of E. coli SoxR indicates that Lys89 and Lys92, located just upstream of dimerization helix 5, are adjacent to [2Fe-2S] (Figure 1-B), yet no electron density has been observed for these two residues in the X ray diffraction data.20 Involvement of Lys residues has been suggested frequently for enzymatic reactions with O2·-. Lys residues are responsible for the high, diffusion-limited rate constant of reactions between superoxide dismutase (SOD) and O2·-.25-27 In addition, the catalytic mechanism for O2·- reduction of superoxide reductase (SOR) is proposed to involve the stabilization of the hydroperoxo intermediate species by Lys.28, 29 For most species of the enteric bacteria, SoxR contains a three-residue hydrophilic motif (Arg127Ser128Asp129) in the vicinity of [2Fe-2S] clusters (Figure 1-B). These residues are not
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conserved in SoxR of other bacteria, including P. aeruginosa (Figure 1-A).21 It has been proposed that the presence of this conserved motif affects the sensitivity of SoxR to redoxcycling compounds.21 Indeed, substitutions to the site in P. aeruginosa SoxR (L125R or L125A) yielded constitutively active variants.21 These species-specific residues may contribute to SoxR sensitivity to redox-active molecules. We further explored the molecular mechanism of O2·- recognition by the SoxR [2Fe-2S] cluster, based on a structure-informed SoxR amino acid sequence alignment. Pulse radiolysis studies of SoxR proteins substituted at K89 and K92 suggest that Lys residues adjacent to [2Fe2S] clusters are critical for this protein’s sensitivity to O2·-. MATERIALS AND METHODS Expression and Purification-The expression plasmids for E. coli30 and P. aeruginosa13 SoxR were transformed into E. coli C41(DE3) and co-expressed with the isc operon for assembly of the Fe-S clusters,31 as described previously. The cells carrying expression plasmids for SoxR were grown at 37 °C in Terrific Broth medium with appropriate antibiotics (50 µg/mL ampicillin and 10 µg/mL tetracycline) and 0.1 mg/ml ferric ammonium citrate. Expression was induced by adding 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at an OD600 ~0.5; subsequently the cells were grown at 18 °C for 24 h. SoxR was purified essentially as described previously.32 SoxR protein samples were purified in their oxidized form, and confirmed as >95% homogeneous by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The concentration of SoxR was determined using an extinction coefficient of 12.7 mM-1 cm-1 at 417 nm.33 Site-directed Mutagenesis-Mutations in the P. aeruginosa or E. coli SoxR genes were generated using the Quick Change mutagenesis kit from Stratagene following manufacture’s recommendations. The presence of the desired mutations was confirmed by DNA sequence
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analysis. The purification procedures for variant proteins were similar to those for wild-type SoxR purification. Materials-Human Cu/Zn SOD, overproduced by E. coli, was kindly provided by Nippon KAYAKU. The enzymic reaction rate constants of SOD were measured by pulse radiolysis method.34 All other reagents were commercially obtained as analytical grade. Pulse Radiolysis-Samples of SoxR for pulse radiolysis were prepared by bubbling a solution containing 20 mM potassium phosphate, 10 mM potassium/sodium tartrate, 0.5 M KCl, and 0.1 M sodium formate (for scavenging OH·) with O2 gas for 2 min, and then adding a concentrated solution of SoxR (1-2 mM). The buffers used were 10 mM acetate buffer (pH 5-6), 10 mM potassium phosphate buffer, or 10 mM potassium borate (pH 8-9). The pH was adjusted with NaOH or HClO4. Pulse radiolysis experiments were performed with a linear accelerator at the Institute of Scientific and Industrial Research, Osaka University.19, 23, 35-37 The pulse width and energy were 8 ns and 27 MeV, respectively. A 1-kW xenon lamp was used as a light source. After passing through an optical path, the transmitted light intensities were analyzed and monitored with a fast spectrophotometric system composed of a Nikon monochromator, an R-928 photomultiplier, and a Unisoku data analysis system. For the time-resolved transient absorption spectral measurement, the monitor light was focused into a quartz optical fiber, which transported the electron pulse induced transmittance changes to a gated-multichannel spectrometer (Unisoku, TSP-601-02). The initial concentration of O2·- generated by pulse radiolysis was 20-50 µM, which was estimated using the relationship, ε260 = 1925 M-1 cm-1.38 The second-order rate constants in the reaction of O2·- with SoxRred were determined from the SOD dose-dependent inhibition in the oxidation of SoxRred.19 Curve-fittings were performed to obtain the data based on the SOD dose-dependent inhibition. The following equations used to the best fits:
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∆A0 and ∆At: absorbance recovery at 420 nm in the absence and presence of SOD, k1 and k2: the rate constants of O2·- with SoxRred and SOD, [SOD] and [SoxRred]: the concentrations of SOD and SoxRred. The ratios of ∆At to ∆A0 were plotted against the concentration of SOD. A rate (k1) for the reaction of with SOD is 2 x 109 M-1 s-1.39 The concentrations of SoxRred were determined by the absorbance changes at 420 nm after pulse radiolysis.
β-Galactosidase assay-β-Galactosidase assays in strain EH46 (∆soxR soxS::lacZ)40 expressing various SoxR proteins was determined as previously described.41 EH46 was kindly provided by Prof.
Bruce
Demple
(Department
of
Genetics
and
Complex
Disease
Harvard School of Public Health). The transformed strains were inoculated into LB medium containing 100 µg of ampicillin/ml and incubated at 37 oC for ~16 h with shaking at 220 rpm. Inocula from the overnight cultures were diluted 100-fold into 3 ml of fresh medium in tubes and incubated at 37 oC for 90 min. PQ was then added to final concentration of 100 µM, and the incubation was continued for 60 min with shaking at 220 rpm. β-Galactosidase activity was assayed by adding o-nitrophenyl-β-D-galactopyranoside after permeabilization of the cell with SDS-chloroform. Spectrophotometric Measurements-Optical absorption spectra were measured with a Hitachi U2900 spectrometer.
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RESULTS
We hypothesized that changing the non-conserved residues in E. coli to those found in P. aeruginosa SoxR might change the O2·- sensitivity of this protein. We individually substituted residues in E. coli SoxR (K89A, K92A, K89AK92A, K89EK92E, K89RK92R, D129A, R127LS128QD129A) and P. aeruginosa SoxR (A87K, A90K, and L125RQ126SA127D) (Figure 1-B). The [2Fe-2S] centers in purified WT SoxR of E. coli and P. aeruginosa SoxR proteins produced visible absorption spectra with peaks 332, 414, 462, and 560 nm.11,
13
Absorption spectra of the purified variants were similar to those of the wild type protein (Figure S1), suggesting that [2Fe-2S] clusters of the variants remained intact. Reaction of O2·- of the Variant SoxR in E. coli. Pulse radiolysis experiments involve the instantaneous generation of hydrated electrons (eaq-). The eaq- reacts with both [2Fe-2S] cluster of SoxRox and O2 to form SoxRred and O2·- as shown in reactions (3) and (4).19
The reduction of the [2Fe-2S] cluster was reflected by a decrease in absorbance at 420 nm. Subsequently, these initial changes in absorbance partially reversed on a time scale of milliseconds (Figures 2). The recovery oxidation was inhibited by addition of Cu/Zn-SOD without affecting the initial rapid decrease. From these results, we concluded that the recovery process reflects the oxidation of SoxRred by O2·- in reaction (1).19 We determined a rate constant of 5 × 108 M-1 s-1 for the reaction of O2- with SoxRred, given the concentration of SOD needed to halfmaximally inhibit oxidation of SoxRred and a rate (k1) for the reaction of O2- with SOD of 2 × 109 M-1 s-1.
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Similar processes were obtained with E. coli SoxR variants (Figure 2). However, the slower millisecond scale changes were different among the variants of E. coli SoxR, whereas the initial rapid changes were not affected. The sensitivity of O2·- of the variants was verified by SOD dose-dependent inhibition. Notably, two amino acid substitutions, K89A and K92A of E. coli SoxR, dramatically affected this protein’s reaction with O2·-. The O2·- sensitivity of the variants is revealed by SOD inhibition. The oxidation of K89AK92A E. coli SoxR was nearly inhibited by 2 µM SOD, whereas this same concentration resulted in ~60 % inhibition in wild SoxR (Figures 2A and B). The Lys substitution led to enhanced SOD inhibition in the order K89A < K92A
