Title: A tyrosine kinase and its activator control the activity of the CtsR heat shock repressor in B. subtilis
Abstract: Artilce15 September 2005free access A tyrosine kinase and its activator control the activity of the CtsR heat shock repressor in B. subtilis Janine Kirstein Corresponding Author Janine Kirstein Ernst Moritz Arndt Universität Greifswald, Institut für Molekulare Mikrobiologie, Greifswald, Germany Freie Universität Berlin, Institut für Biologie-Mikrobiologie, Berlin, Germany Search for more papers by this author Daniela Zühlke Daniela Zühlke Ernst Moritz Arndt Universität Greifswald, Institut für Molekulare Mikrobiologie, Greifswald, Germany Search for more papers by this author Ulf Gerth Ulf Gerth Ernst Moritz Arndt Universität Greifswald, Institut für Molekulare Mikrobiologie, Greifswald, Germany Search for more papers by this author Kürşad Turgay Kürşad Turgay Freie Universität Berlin, Institut für Biologie-Mikrobiologie, Berlin, Germany Search for more papers by this author Michael Hecker Michael Hecker Ernst Moritz Arndt Universität Greifswald, Institut für Molekulare Mikrobiologie, Greifswald, Germany Search for more papers by this author Janine Kirstein Corresponding Author Janine Kirstein Ernst Moritz Arndt Universität Greifswald, Institut für Molekulare Mikrobiologie, Greifswald, Germany Freie Universität Berlin, Institut für Biologie-Mikrobiologie, Berlin, Germany Search for more papers by this author Daniela Zühlke Daniela Zühlke Ernst Moritz Arndt Universität Greifswald, Institut für Molekulare Mikrobiologie, Greifswald, Germany Search for more papers by this author Ulf Gerth Ulf Gerth Ernst Moritz Arndt Universität Greifswald, Institut für Molekulare Mikrobiologie, Greifswald, Germany Search for more papers by this author Kürşad Turgay Kürşad Turgay Freie Universität Berlin, Institut für Biologie-Mikrobiologie, Berlin, Germany Search for more papers by this author Michael Hecker Michael Hecker Ernst Moritz Arndt Universität Greifswald, Institut für Molekulare Mikrobiologie, Greifswald, Germany Search for more papers by this author Author Information Janine Kirstein 1,2, Daniela Zühlke1, Ulf Gerth1, Kürşad Turgay2 and Michael Hecker1 1Ernst Moritz Arndt Universität Greifswald, Institut für Molekulare Mikrobiologie, Greifswald, Germany 2Freie Universität Berlin, Institut für Biologie-Mikrobiologie, Berlin, Germany *Corresponding author. Freie Universität Berlin, Institut für Biologie-Mikrobiologie, Königin-Luise-Str. 12-16, 14195, Berlin, Germany. Tel.: +49 30 838 53111; Fax: +40 30 838 53118; E-mail: [email protected] The EMBO Journal (2005)24:3435-3445https://doi.org/10.1038/sj.emboj.7600780 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The soil bacterium Bacillus subtilis possesses a fine-tuned and complex heat stress response system. The repressor CtsR, whose activity is regulated by its modulators McsA and McsB, controls the expression of the cellular protein quality control genes clpC, clpE and clpP. Here, we show that the interaction of McsA and McsB with CtsR results in the formation of a ternary complex that not only prevents the binding of CtsR to its target DNA, but also results in a subsequent phosphorylation of McsB, McsA and CtsR. We further demonstrate that McsB is a tyrosine kinase that needs McsA to become activated. ClpC inhibits the kinase activity of McsB, indicating a direct role in initiating CtsR-controlled heat shock response. Interestingly, the kinase domain of McsB is homologous to guanidino phosphotransferase domains originating from eukaryotic arginine and creatine kinases. Mutational analysis of key residues of the guanidino kinase domain demonstrated that McsB utilizes this domain to catalyze the tyrosine phosphorylation. McsB represents therefore a new kind of tyrosine kinase, driven by a guanidino phosphotransferase domain. Introduction The Gram-positive soil bacterium Bacillus subtilis can respond to various and multiple changes of its natural environment. The various cellular stress response systems of B. subtilis enabling this fast adaptation serve as a general model system for regulatory circuits, which include, for example, two-component systems, alternative sigma factors and regulated proteolysis. For one of these systems, the heat shock response of B. subtilis, it could be demonstrated that it is regulated by at least five different mechanisms. The major regulatory proteins of four of these classes were identified and characterized. Class I genes are regulated primarily by the repressor HrcA, the alternative sigma factor σB controls the class II genes, the repressor CtsR controls the class III genes and the two-component system CssRS controls the class V genes (Darmon et al, 2002; Schumann et al, 2002). The proteins we investigated in this study, McsB and McsA, encoded by class III heat shock genes are directly involved in the regulation of class III heat shock response. The major regulator CtsR is a dimeric repressor, which binds to a highly conserved heptanucleotide direct repeat, located upstream of clpP, clpE and the clpC operon (Derre et al, 1999a, 1999b; Krüger and Hecker, 1998). The regulation of the CtsR regulon is thought to be based on maintaining a basal steady-state level of CtsR at 37°C and that at elevated temperatures a rapid degradation of the repressor by ClpCP occurs (Krüger et al, 2001). All the genes of the clpC operon, which consist of ctsR, mcsA, mcsB and clpC, are involved in the regulation of the activity of CtsR, because McsA and McsB act as modulators of CtsR (Krüger et al, 2001). McsB, a putative kinase was shown to repress the DNA-binding ability of CtsR and it was further proposed that McsB could modify CtsR to target it for degradation by ClpCP. Such an McsB-dependent modification of CtsR was detected by an in vivo approach, suggesting a possible phosphorylation of the repressor. However, a kinase activity of McsB was not demonstrated yet. Interestingly, McsB contains a domain that is highly conserved among ATP:guanidino phosphotransferases (referred to as guanidino kinases) (Krüger et al, 2001). This domain is used by the phosphagen kinase family and catalyzes the phosphorylation of guanidino molecules such as arginine or creatine, serving as 'energy-storage' in maintaining energy homeostasis by buffering cellular ATP concentrations of cells of higher eukaryotes, which have to utilize high amounts of ATP (Ellington, 2001). In this study, the precise role of both modulators of CtsR was investigated. Therefore, we established an in vitro phosphorylation assay and could demonstrate a kinase activity of McsB. Surprisingly, the characterization of the phosphoamino acids and mutational analysis of McsA and McsB revealed phosphorylation on tyrosines. Tyrosine phosphorylation was considered to be restricted to eukaryotes until phosphotyrosine kinase (PTK) activity could be demonstrated in Escherichia coli (Manai and Cozzone, 1982). So far, bacterial PTKs were found to be involved in the regulation of the synthesis of exopolysaccharides in B. subtilis and other bacteria (Morona et al, 2000; Mijakovic et al, 2003) and possibly in the regulation of the heat shock response in E. coli (Klein et al, 2003). The tyrosine kinase activity of McsB required the activation by McsA and resulted in phosphorylation of both McsA and McsB. We could further show that CtsR is a bona fide phosphorylation substrate of McsB and that the kinase activity of McsB is antagonized by ClpC, indicating a direct role of this phosphorylation cascade in initiating CtsR-controlled heat shock response. Results and discussion McsB is a protein kinase that is stimulated by McsA and phosphorylates CtsR The amino-acid (aa) sequence of McsB contains a highly conserved domain with similarity to guanidino kinases (kinase domain aa 119–253). Furthermore, the heat shock-induced and McsB-dependent occurrence of an additional acidic charged subspecies of CtsR, detected by two-dimensional gel analysis, suggested modification of CtsR by phosphorylation (Krüger et al, 2001). In order to experimentally examine a possible kinase activity of McsB, we used the purified components to establish an in vitro phosphorylation assay, using radioactively labeled [γ-32P]ATP. The results of this assay depicted in Figure 1 demonstrate that McsB alone, unlike McsA or CtsR, appeared to be phosphorylated at a very low level (Figure 1A, lane 2) when incubated with the labeled ATP, suggesting a low-level autophosphorylation activity of McsB. Addition of equal amounts of McsA stimulated this activity by several orders of magnitude and led to the concurrent phosphorylation of McsA (Figure 1A, lane 6). Analysis of the time course of this reaction revealed that a maximal level of phosphorylation for both proteins was achieved after 20 min and remained stable for at least an additional 60 min (Figure 1B, and data not shown). The addition of equimolar amounts of CtsR to this in vitro phosphorylation assay resulted in the immediate phosphorylation of CtsR, but only in the presence of both McsA and McsB (Figure 1A, lanes 7 and 3–5). We titrated the amount of CtsR and observed that the phosphorylation of CtsR became saturated at a ratio of two CtsR per McsA/McsB (data not shown). It was previously observed that a dimer of CtsR is the active species (Derre et al, 1999a, 1999b, 2000; Krüger and Hecker, 1998). Figure 1.Characterization of the phosphorylation activity of McsB. (A) McsB exhibits a weak autophosphorylation activity, which is strongly stimulated by McsA, and phosphorylates McsA and CtsR. McsB, McsA and CtsR was incubated with [γ-32P]ATP for 20 min and subsequently analyzed by SDS–PAGE and autoradiography (as indicated below the autoradiogram). The position of McsB, McsA and CtsR is indicated on the right side. The star indicates a low abundant contaminant of the McsB purification from E. coli, which possesses an autophosphorylation activity independent of McsB. (B) Quantitative analysis of the time course of the phosphorylation of McsB and McsA. Filled squares represent the relative phosphorylation of McsB and filled circles McsA. The inset depicts the autoradiogram of the time course of the phosphorylation level of McsA and McsB. McsA (1 μM) and McsB (1 μM) were incubated with [γ-32P]ATP and samples were withdrawn at the indicated time points, analyzed and quantified with a phosphorimager. Download figure Download PowerPoint We conclude from these results that McsA acts as a specific activator necessary for the full kinase activity of McsB, resulting in the phosphorylation of McsB, McsA and subsequently CtsR. Interaction of McsB with McsA and CtsR To allow phosphorylation of McsA, McsB and CtsR, an interaction between McsA, McsB and CtsR must occur. To investigate this complex in more detail, we used two approaches. First, as an in vivo approach, co-immunoprecipitation experiments were performed with either McsA, McsB or CtsR antibodies immobilized on protein A-coated magnetic beads and lysates were prepared from wild-type (wt), ΔmcsA, ΔmcsB or ΔctsR mutant cells, which were grown at 37°C or heat shocked at 50°C. Subsequently, a Western blot was performed to analyze whether McsA, McsB or CtsR was co-immunoprecipitated from the lysates. The experiment shown in Figure 2A demonstrates that using McsA antibodies, capture of CtsR by McsA was possible only when wt lysate and not lysate prepared from ΔmcsB cells was used (Figure 2A). This indicated that the presence of McsB is necessary for an interaction of McsA with CtsR. In a pull-down experiment using McsB antibodies, CtsR and McsA co-precipitated together with McsB. CtsR could also be detected in the same experiment using a lysate prepared from ΔmcsA cells (Figure 2B), which demonstrated that the interaction of McsB with CtsR was independent of McsA. Using CtsR antibodies, McsB co-precipitated in a wt as well as in a ΔmcsA extract, whereas McsA co-precipitated only in lysates of wt but not of ΔmcsB cells (Figure 2C). In summary, these experiments suggested that McsA and CtsR were able to bind simultaneously to McsB and that the interaction of CtsR with McsA proceeded via McsB. Figure 2.Analysis of the interaction between McsA, McsB and CtsR. (A–C) Pull-down experiments using protein A-coupled McsA (A), McsB (B) and CtsR antibodies (C), with lysates prepared from B. subtilis 168 (wt), ΔctsR, ΔmcsA or ΔmcsB strains (as indicated above). Co-precipitated proteins were analyzed by SDS–PAGE and subsequent Western blotting with the respective antisera (indicated on the right). (D) Interaction of McsB with McsA and CtsR by SPR. The binding response is measured in resonance units (RU). McsB was immobilized on a CM5 chip and McsA (0.8 μM) and CtsR (0.8 μM) (as indicated in the sensorgram) were passed over the chip surface as analytes. Download figure Download PowerPoint The ability of McsB to interact directly with either McsA or CtsR was confirmed in vitro using surface plasmon resonance (SPR) with a BIAcore instrument. As depicted in Figure 2D, both McsA and CtsR showed interaction with McsB. CtsR, whose binding to McsB was about five times stronger, also appeared to have a very low off-rate compared to McsA binding to McsB. These protein interaction experiments demonstrated the ability of McsB to bind directly to CtsR and to McsA, and the co-immunoprecipitation (co-IP) experiments strongly suggested that McsB was located at the center of a ternary complex of McsA–McsB–CtsR. This implies that the observed induction of the McsB kinase activity by McsA, which resulted in the concurrent phosphorylation of CtsR (Figure 1A), takes place in this ternary complex. Phosphorylation occurs on tyrosine residues To gain insight into the nature of the phosphorylation events, catalyzed by McsB and McsA, we wanted to determine what kind of amino acid was phosphorylated. First, we examined the stability of the phosphorylation of McsA, McsB and CtsR at high temperature (95°C), acidic (HCl, 1 M) and basic (NaOH, 1 M) conditions. Our results demonstrated that the phosphorylation of McsA, McsB and CtsR was stable under all these conditions (Figure 3A). The observed stability under acidic and heat conditions is consistent with hydroxyamino acid phosphorylation and since only phosphotyrosine residues resist high pH, these experiments suggested tyrosines as phosphorylation sites (Duclos et al, 1991). Figure 3.Analysis of the phosphoamino acid. (A) Autoradiogram of the chemical stability of McsA, McsB and CtsR. All three proteins (1 μM) were incubated with [γ-32P]ATP for 20 min and subsequently treated with HCl, NaOH or boiled at 95°C (as indicated below) for an additional 10 min. The position of McsA, McsB and CtsR is depicted on the right. (B) Autoradiogram of the analysis of the phosphoamino acid by two-dimensional thin-layer chromatography of hydrolyzed McsA∼P and McsB∼P. An overlay with the position of the standard phosphoamino acids, P∼Ser, P∼Thr and P∼Tyr (∼20 μg), which was analyzed by ninhydrin staining, is indicated by dotted circles. (C) Dephosphorylation of McsA∼P and McsB∼P by YwlE. McsA and McsB were preincubated with [γ-32P]ATP for 20 min. Then, 1 μM YwlE was added at time point 0 (lanes 5–8) or not added (lanes 1–4) and the reaction was followed for another 60 min. Samples were withdrawn at the indicated time points and analyzed by SDS–PAGE and autoradiography. The relative position of McsB∼P and McsA∼P is depicted on the left. YwlE did not alter the serine phosphorylation of RsbV by RsbW. RsbW and RsbV were incubated with [γ-32P]ATP as described above and the phosphorylation was followed in the presence and absence of YwlE (lanes 9–14). (D) Dephosphorylation of CtsR by YwlE. McsA, McsB and CtsR (1 μM) were incubated with [γ-32P]ATP for 20 min, followed by the addition of YwlE (as indicated below), and analyzed as described above. The relative position of McsB∼P, McsA∼P and CtsR∼P is depicted on the left. Download figure Download PowerPoint To verify this assumption, we used two-dimensional thin-layer chromatography to analyze the phosphoamino acid (Mijakovic et al, 2003). The migration pattern of the radiolabeled hydrolysis products was compared with phosphoamino acid standards P∼Ser, P∼Thr and P∼Tyr. As shown in Figure 3B, radioactive products of hydrolyzed McsA and McsB comigrated with the P∼Tyr standard. The chemical stability and the phosphoamino acid analysis by two-dimensional thin-layer chromatography demonstrated that McsB and McsA became phosphorylated at tyrosines. YwlE, a tyrosine phosphatase, dephosphorylates McsA, McsB and CtsR It was previously proposed by Kobayashi and colleagues that YwlE, a protein with homology to low molecular weight tyrosine phosphatases, carrying the conserved active site signature motif, CTGNTCRS/T (Zhang et al, 1995), could act as counterpart to McsB (Schumann et al, 2002). In addition, Mijakovic et al (2003) reported that YwlE could dephosphorylate the autophosphorylated tyrosine kinase, YwqD, and two proteins phosphorylated at tyrosines, [P-Tyr]-YwqF and [P-Tyr]-TuaD. We cloned, expressed and purified YwlE and tested its effect on the in vitro phosphorylation of McsA and McsB. Both proteins were preincubated for 20 min with [γ-32P]ATP to gain the maximal phosphorylation state (Figure 1B), YwlE was added and samples were withdrawn subsequently following the indicated time course (Figure 3C). Compared to the control reaction in the absence of YwlE, an immediate loss of the phosphorylation signals of McsA and McsB was observed (Figure 3C). The phosphatase activity of YwlE could also be demonstrated for CtsR (Figure 3D). Furthermore, the presence of YwlE did not alter the Ser56 phosphorylation of RsbV by RsbW, which served as a control and demonstrated the P∼Tyr specificity of YwlE (Figure 3C). We also purified YwqE, which is the cognate tyrosine phosphatase of YwqD, YwqF and TuaE (Mijakovic et al, 2003), and YfkJ, the closest paralog (30% identity) of YwlE. Unlike YwlE, neither of these two phosphatases interfered with the phosphorylation state of McsA and McsB (data not shown). The dephosphorylation of McsA, McsB and CtsR by the tyrosine phosphatase YwlE further supported our previous findings (Figure 3A and B), that McsB, its activator protein McsA and their substrate-protein CtsR are phosphorylated at tyrosine residues. Mapping of the phosphorylation sites within McsB and McsA The most conserved tyrosine residues of McsB, among those Gram-positive bacteria with a low GC content that encode for an McsB ortholog, are located within the kinase domain (aa 119–253; Y155, Y163 and Y210) (Krüger et al, 2001). Nevertheless, we substituted all eight tyrosine residues of McsB against its closest structural homolog, phenylalanine. Figure 4A depicts the result of the kinase assay of McsA with wt McsB as well as all tyrosine point mutants of McsB. No phosphorylation of either McsA or McsB in the assay could be detected for McsBY155F and only a very weak signal could be detected for McsBY210F. This demonstrated that both tyrosine residues necessary for the kinase activity of McsB are possible targets for phosphorylation. Figure 4.Mapping of the phosphorylation sites within McsB and McsA. (A) Autoradiogram of a phosphorylation assay of McsA with McsB and all YF point mutants of McsB (as indicated above). The position of McsB∼P and McsA∼P is depicted on the left. (B, C) The phosphorylation activity of McsB could not be complemented in trans. McsBY155F or McsB210F (B) and McsBY155E or McsBY210E (C), respectively, either alone or together (as indicated above the autoradiograms), were incubated with McsA and [γ-32P]ATP for 20 min and analyzed by SDS–PAGE and autoradiography. The position of McsB∼P and McsA∼P is depicted on the left. (D) The McsA phosphorylation did not result from a Pi transfer from McsB∼P. McsB (10 μM) was incubated with [γ-32P]ATP for 30 min, purified from free [γ-32P]ATP and subsequently incubated with McsA (10 μM). Samples were withdrawn immediately after the addition of McsA (0 min) and 30 min as well as 60 min later (as indicated above the autoradiogram) and analyzed by SDS–PAGE and autoradiography. The position of McsB∼P and McsA is depicted on the left. (E) McsA is phosphorylated at the tyrosines Y40 and Y103. Autoradiogram of the phosphorylation assay of McsB with McsA and both YF point mutants of McsA (as indicated above) is depicted. The position of McsB∼P and McsA∼P is depicted on the left. (F) The acidic charge of the McsA phosphorylation could be mimicked by replacement of Y against E and thereby recovering the kinase activity of McsB. McsB was incubated with each of McsA, McsAY40E and McsAY103E (as indicated above) in the presence of [γ-32P]ATP for 20 min and analyzed by SDS–PAGE and autoradiography. The position of McsB∼P and McsA∼P is depicted on the left. Download figure Download PowerPoint These results implicated that an intramolecular phosphate transfer, as recently discussed for the KaiC phosphorylation (Xu et al, 2004), could occur in McsB, although it cannot be excluded that the protein kinase gains its activity in a two-step mechanism as shown for the E. coli PTK Wzc (Grangeasse et al, 2002). To test such a two-step process, which requires an initial phosphorylation to activate the kinase, we created Y155E and Y210E point mutants of McsB to mimic phosphorylation by an acidic charge of glutamate (Gryz and Meakin, 2003). As shown in Figure 4C, no kinase activity could be detected for these mutants as well as for the YF substitutions of Y155 and Y210 (Figure 4A). Since no phosphorylation appeared even in the YE variants of McsB, we conclude that McsB does not employ an intrinsic two-step mechanism as described for Wzc (Grangeasse et al, 2002). In gel filtration experiments, McsB appeared to run as a monomer (data not shown), but nevertheless we also tested a 1:1 mixture of both single mutant proteins, YF as well as YE substitutions of Y155 and Y210, in the kinase assay. But no phosphorylation signal appeared, suggesting that a missing tyrosine could not be complemented in trans by a tyrosine in another McsB molecule (Figure 4B and C). These results support our first assumption of an intramolecular phosphate shuttle between the two tyrosines in McsB. We were interested in whether the McsA phosphorylation is achieved by an intermolecular phosphate transfer from McsB∼P and is thereby a part of the putative phosphate shuttle. We addressed this issue by incubation of McsA with purified [32P]Tyr-McsB, where free [γ-32P]ATP was removed. As depicted in Figure 4D, no McsA phosphorylation signal could be detected in an autoradiogram, suggesting that the McsA phosphorylation did not result from a phosphate transfer from McsB∼P but rather utilized the γ-phosphate of a new ATP molecule. McsA is characterized by its two Zn-binding motifs in the N-terminus and a C-terminal uvr domain (amino-acid residues 139–174). Deletion of this C-terminal domain did not abolish the phosphorylation signal, demonstrating that phosphorylation takes place in the N-terminus (data not shown). Indeed, using single phenylalanine substitutions of the N-terminal-localized two tyrosines of McsA (McsAY40F and McsAY103F), no phosphorylation of McsB or McsA was observed in the kinase assay (Figure 4E). This raised the question whether only the phosphorylated state of McsA stimulates the kinase activity of McsB. To test this hypothesis, we substituted the tyrosine residues of McsA against glutamate to mimic the acidic charge of phosphorylation. As depicted in Figure 4F, both single tyrosine to glutamate (McsAY40E, McsAY103E) substitutions, unlike the previously described tyrosine to phenylalanine substitutions (McsAY40F, McsA103F), were able to activate McsB, resulting in the phosphorylation of McsB as well as the second, not altered, tyrosine residue of McsA. This suggested that both tyrosines of McsA are not only required for phosphorylation of McsA and the activation of McsB but were both themselves phosphorylated. In summary, these experiments demonstrate that the tyrosines 155 and 210 of McsB and the tyrosines 40 and 103 of McsA could be phosphorylation targets and that they are essential for the tyrosine kinase activity of McsB and its activation by McsA. Moreover, our experiments indicate that the phosphorylation of McsB resulted from an intramolecular phosphate transfer between both phosphorylation sites, Y155 and Y210, whereas no intermolecular phosphate transfer from McsB∼P to McsA was detected. McsB-mediated release of CtsR from DNA is enhanced in the presence of McsA and ATP Our results indicated that McsA could form a ternary complex with McsB and CtsR and concurrently activated the kinase activity of McsB. Therefore, we investigated the influence of McsA and McsB on the DNA-binding activity of CtsR in more detail. The repression of the class III heat shock genes is based on the binding of the dimeric CtsR to a heptanucleotide direct repeat, which is located in varying copies within the promoter regions of clpC, clpE and clpP. To monitor the DNA-binding ability of CtsR, we amplified the promoter region of clpC, which contains two CtsR-binding sites. As already shown, McsB antagonizes the CtsR–DNA interaction (Krüger et al, 2001). However, titration of both modulators allowed a more precise analysis. Increasing amounts of McsB prevented DNA binding by CtsR (Figure 5A). This McsB-dependent inhibition of CtsR binding was enhanced by McsA, in the presence of ATP, reducing the McsB level, which is required for its inhibiting effect, by half (Figure 5A). ATP depletion or replacement by the slow hydrolyzable analog, ATPγS, weakens the influence of McsA (data not shown). This McsA activation appears to be coupled to the presence of ATP, indicating phosphorylation of McsB. Figure 5.Modulation of the CtsR–DNA interaction by McsA and McsB measured by DNA gel retardation. (A) Influence of McsA, McsB and ATP on the DNA-binding ability of CtsR. CtsR (1 μM) was incubated with McsA (1 μM), 1 mM ATP and increasing concentrations of McsB (0–1 μM) (as indicated below the gel). DNA-binding analysis was initiated by addition of the promoter fragment. The DNA retardation was analyzed by ethidium bromide staining of the native gel. (B) Influence of the McsBY155E mutation on the CtsR–DNA interaction. McsBY155E, a variant of McsB, which carries an acidic charge at one phosphorylation site, was tested for its influence on the CtsR activity as described above. Download figure Download PowerPoint To verify the assumption that phosphorylated McsB exhibits a higher affinity toward CtsR, we tested the phosphorylation mimicking McsB mutant, McsBY155E, in the gel retardation assay. As depicted in Figure 5B, McsBY155E could diminish the CtsR–DNA interaction alone as efficient as wt McsB in the presence of McsA and ATP. However, since McsB is able to inhibit DNA binding of CtsR on its own, phosphorylation of CtsR cannot be a prerequisite for its release. It seems more likely to assume a higher affinity of McsB for CtsR in a phosphorylated state, either alone or together with McsA. In summary, these results demonstrate that phosphorylated McsB, activated by McsA, is a stronger inhibitor of the CtsR repressor than unphosphorylated McsB. A structural model of the guanidino kinase domain of McsB A structural model of the McsB kinase domain would be valuable to understand the role of the guanidino kinase domain and the tyrosines in this new tyrosine kinase. Two of the eight tyrosines of McsB are necessary for the kinase activity of McsB. They are conserved in all McsB homologs and located within the kinase domain, but they are not conserved in AK/CK (Figure 6A). This suggests that these tyrosines, which are possible phosphorylation targets, are not necessarily key residues of the catalytic mechanism of guanidino kinase domains. A number of creatine and arginine kinases bearing the guanidino kinase domain were crystallized and their structures solved (Fritz-Wolf et al, 1996; Zhou et al, 1998). Based on this, SwissModel (http://swissmodel.expasy.org//SWISSMODEL.html) a fully automated protein structure homology modeling server, could successfully be used to obtain a structural model of the kinase domain of McsB (Figure 6A). The program based that model of the McsB kinase domain (aa 119–253) on the homologous domain of different arginine and creatine kinase structures deposited in PDB (1bg0, 1m15, 1p50, 1p52, 1qk1, 1rl9). Figure 6A shows an alignment of the McsB kinase domain with the guanidino kinase domain of the AK from Limulus polyphemus (PDB code 1bg0), whose previously determined crystal structure served also as model template (Zhou et al, 1998). The activity of AK is controlled by a conformational switch, upon binding of substrate and ATP ('induced fit'), and relies on several key residues and structural elements. These include (in the AK nomenclature) the essential C271, the highly conserved NEED segment (residues 223–227), the flexible loop (aa 309–320) with E314 and the arginines R229, R280 and R309. As depicted in the alignment (Figure 6A), all these key residues are conserved in McsB. Y210 of McsB is located within the flexible loop region (aa 309–320). For the glutamate (E314) of this flexible loop region, it was demonstrated that it interacts together with a cysteine (C271) and a glutamate (E225 of the NEED motif) with the substrate arginine upon binding of ATP. All three amino acids thereby stabilize and orient the substrate arginine in the transition state of the phosphate transfer reaction of AK. The second tyrosine of McsB, Y155, is located on the other side of the ATP-binding domain with C271 and the NEED