Báo cáo khoa học: Identification, characterization and activation mechanism of a tyrosine kinase of Bacillus anthracis docx

Identification, characterization and activation mechanism of a tyrosine kinase of Bacillus anthracis Abid R. Mattoo, Amit Arora, Souvik Maiti and Yogendra Singh Institute of Genomics and Integrative Biology, Delhi, India Serine, threonine and tyrosine protein kinases represent an emerging concept in prokaryotic signalling, and have been implicated in a variety of control mechanisms, including stress responses, developmental processes and pathogenicity [1,2]. Among various types of protein phosphorylation in bacteria, least is known about tyro- sine phosphorylation and its physiological role [3]. These protein-tyrosine kinases possess conserved nucle- otide-binding motifs known as Walker A, A¢ and B, with some exceptions [4]. The majority of the bacterial tyrosine kinases possess a transmembrane domain and an intracellular catalytic domain [3,4]. These two domains are present either on a single polypeptide (pro- teobacteria and actinobacteria), or exist as separate entities. Moreover, it has been reported in firmicutes that transmembrane protein act as a modulator and influences the kinase activity of the catalytic domain [4]. McsB is a unique tyrosine kinase of Bacillus subtilis that contains a eukaryotic-like guanidino-phospho- transferase domain for its kinase activity. McsB, together with McsA, is known to modulate the activity of the repressor of the class III heat shock genes (CtsR) in B. subtilis. McsB exhibits autophosphorylation activ- ity, a common feature of all tyrosine kinases. However, the maximal kinase activity of McsB was observed only in the presence of McsA. The presence of McsA not only enhanced the autophosphorylation activity of McsB, but also resulted in phosphorylation of McsA. It has been shown that multiple sites are phosphorylated on both McsB and McsA [5]. However, the mechanism of enhanced phosphorylation of McsB in the presence of McsA is not understood. Bacterial tyrosine kinases have been found to control exopolysaccharide production in both Gram-positive Keywords Bacillus anthracis; ITC; McsB; SPR; tyrosine kinase Correspondence S. Maiti and Y. Singh, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007, India Fax: +91 11 27667471 Tel: +91 11 27666156 E-mail: souvik@igib.res.in; ysingh@igib.res.in (Received 19 August 2008, revised 13 October 2008, accepted 17 October 2008) doi:10.1111/j.1742-4658.2008.06748.x Bacillus subtilis has three active tyrosine kinases, PtkA, PtkB and McsB, which play an important role in the physiology of the bacterium. Genome sequence analysis and biochemical experiments indicated that the ortholog of McsB, BAS0080, is the only active tyrosine kinase present in Bacillus anthracis. The autophosphorylation of McsB of B. anthracis was enhanced in the presence of an activator protein McsA (BAS0079), a property similar to that reported for B. subtilis. However, the process of enhanced phos- phorylation of McsB in the presence of McsA remains elusive. To under- stand the activation mechanism of McsB, we carried out spectroscopic and calorimetric experiments with McsB and McsA. The spectroscopic results suggest that the binding affinity of Mg-ATP for McsB increased by one order from 10 3 to 10 4 in the presence of McsA. The calorimetric experi- ments revealed that the interaction between McsB and McsA is endother- mic in nature, with unfavourable positive enthalpy (DH) and favourable entropy (DS) changes leading to an overall favourable free energy change (DG). Kinetics of binding of both ATP and McsA with McsB showed low association rates (k a ) and fast dissociation rates (k d ). These results suggest that enhanced phosphorylation of McsB in the presence of McsA is due to increased affinity of ATP for McsB. Abbreviations HK, histidine kinase; ITC, isothermal titration calorimetry; PtkA, protein tyrosine kinase A; PtkB, protein tyrosine kinase B; RU, response units; SPR, surface plasmon resonance. FEBS Journal 275 (2008) 6237–6247 ª 2008 The Authors Journal compilation ª 2008 FEBS 6237 and Gram-negative bacteria. Exopolysaccharides play an important roles in bacterial virulence, suggesting a role for tyrosine kinases in bacterial pathogenesis [6]. In addition, tyrosine kinases have been found to phosphorylate RNA polymerase sigma factors in Escherichia coli [7], UDP-glucose dehydrogenases in E. coli and B. subtilis [8,9] and single-stranded DNA- binding proteins in B. subtilis [10]. Bacillus anthracis, the causative agent of anthrax, is a Gram-positive, spore-forming bacterium. Many of the sensor kinases involved in the initiation of sporulation in B. anthracis are inactive [11]. Comparison of the two-component system of B. anthracis with those of other members of the Bacillus cereus group shows that B. anthracis appears to lack some of the important histidine kinases (HKs) and response regulators, and contains many truncated, possibly nonfunctional, HK and response regulator genes [12]. In the absence of sev- eral important HKs, it is possible that serine ⁄ threonine and tyrosine kinases may have an important role to play in the physiology and pathogenesis of B. anthracis. In this article, for the first time we show the presence of an active tyrosine kinase in B. anthracis. We also show the mechanism by which the kinase activity of B. anthracis is enhanced by the modulator protein. Results and Discussion Distribution of tyrosine kinases in B. anthracis Earlier studies have shown the presence of six putative tyrosine kinases in B. subtilis, which include YwqD, YveL, SojA, SalA, MinD and McsB [5,9,10,13–16]. However, only three proteins, YwqD [protein tyrosine kinase A (PtkA)], YveL [protein tyrosine kinase B (PtkB)] and McsB have been reported to possess tyro- sine kinase activity. These tyrosine kinases have been shown to regulate various physiological processes in B. subtilis [5,9,10,13–15]. SojA and MinD have a con- siderably shorter N-terminal region preceding the Walker motif A than the region present either in PtkA or PtkB, and were unable to autophosphorylate in vitro [9]. SojA and MinD have been implicated in chromosome partitioning and cell division [16,17]. SalA was found to negatively regulate expression of ScoC [14], a transcriptional regulator participating in the control of peptide transport and sporulation initia- tion. All three proteins (MinD, SojA and SalA) lack a transmembrane activator protein in their vicinity, which is required for the activity of tyrosine kinases in firmicutes [4]. A blastp search was performed in the B. anthracis nonredundant protein sequence database (NCBI, NIH) to identify the orthologs of each of these proteins in B. anthracis. To resolve instances where more than one protein was obtained by the blast search, auxiliary criteria such as symmetrical best hit (SymBet) and conservation of order of genes (synteny) were used to define an ortholog [18,19]. A blast search using the PtkA and PtkB protein sequences against the B. anthracis database revealed that the orthologs of these kinases are absent in B. anthracis. Interestingly, using a similar strategy to identify the orthologs of the modulators (YwqC and YveK) of these kinases, we found that their corresponding ortho- log BAS1491 (Wzz) was present in B. anthracis, and showed significant similarity of 51% with YwqC and 48% with Yvek. The comparison of the genes around the modulators yvek (wzz) and BAS1491 (wzz) (Fig. S1A) revealed that, in place of the tyrosine kinase PtkB (YveL), B. anthracis has genes encoding two hypothetical proteins, BAS1492 and BAS1493. The other genes encompassing gene locus (BAS1492 + BAS1493) and ptkB are conserved (genes in both the organisms code for proteins involved in cell wall ⁄ mem- brane ⁄ envelope biogenesis). The presence of these hypothetical proteins upstream of Wzz in all strains of B. anthracis precludes the possibility of sequencing error. The gene locus containing BAS1492 and BAS1493 shows high sequence divergence from ptkB at the nucleotide level in comparison to the other neighbouring genes. It suggests that these hypothetical proteins (BAS1492 and BAS1493) may have been formed due to recombination, insertion or deletion in the nucleotide sequence of genes encoding PtkB-like kinases and evolved to its current nonfunctional forms. The orthologs of SalA (BAS0147 and BAS3357, show- ing similarities of 85% and 65%, respectively), MinD (BAS4346, showing a similarity of 90%) and SojA (BAS5333 and BAB82448, showing similarities of 92% and 47%, respectively) are present in B. anthracis. BAB82448 is present on pathogenic plasmid pX02. The comparison of genes around orthologs of SalA, MinD and SojA in B. anthracis revealed that these orthologs also lack an activator transmembrane protein in their vicinity, like their counterparts in B. subtilis. Thus, these proteins also lack tyrosine kinase activity. However, the blast search of McsB and its activator McsA in the B. anthracis database revealed two orthologs, BAS0080 (81% similarity) and BAS0079 (69% similarity). The alignment of McsB of B. anthracis (BAS0080) with McsB of B. subtilis and a few other organisms is depicted in Fig. S1B. The align- ment suggests that McsB of B. anthracis possesses all the important residues needed for ATP binding and hydrolysis that are present in its counterpart in B. subtilis. These conserved amino acids in McsB of McsB of Bacillus anthracis A. R. Mattoo et al. 6238 FEBS Journal 275 (2008) 6237–6247 ª 2008 The Authors Journal compilation ª 2008 FEBS B. anthracis include Glu120, Glu121 (NEED motif) and Glu212, the catalytic Cys167, and the positively charged Arg125, Arg176 and Arg207 (Figs S1B and S4). The phosphorylation sites Tyr155 and Tyr210 are also conserved in McsB of B. anthracis. Thus, our study suggests that orthologs of two important tyro- sine kinases, PtkA and PtkB, are absent in B. anthra- cis, and that McsB is the only active tyrosine kinase present in this organism. Autophosphorylation of McsB of B. anthracis The autophosphorylation activity of McsB of B. anthr- acis was evaluated as described in Experimental proce- dures. McsB showed autophosphorylation activity that was enhanced by several orders of magnitude upon the addition of McsA (Fig. 1A). In addition to having autophosphorylation activity, McsB also phosphory- lated McsA (Fig. 1A). To determine the phosphory- lated residues, McsB and McsA were incubated either in 1 m HCl or 1 m KOH or incubated at high temper- ature (95 °C) after phosphorylation reactions. The results suggest that the phosphorylation of McsA and McsB was stable under these conditions (Fig. S2). Earlier studies have shown that phosphorylated resi- dues of tyrosine kinases are stable under acidic and alkaline conditions and at high temperature [5], sug- gesting that both McsB and McsA of B. anthracis are phosphorylated on the tyrosine residue. Moreover, mutation of either of the two conserved tyrosine resi- dues, Tyr155 (McsBY155F) and Tyr210 (McsBY210F), resulted in loss of the autophophorylation activity (Fig. 1B). The observed changes in the activity of mutants could be due to alterations in the secondary structure of the proteins. The secondary structures of wild-type and mutant proteins were monitored by CD spectroscopy and represented as observed ellipticity. The CD spectra of wild-type and mutant proteins revealed no significant changes in secondary structure, indicating that loss of activity was not due to struc- tural perturbation (Fig. S3). These observations suggest that phosphorylation of McsB could possibly be due to an intramolecular phosphate transfer between both phosphorylation sites (Tyr155 and Tyr210), as shown earlier for McsB of B. subtilis [5]. These results imply that McsB of B. anthracis is a tyro- sine kinase and that Tyr155 and Tyr210 are the sites of autophosphorylation. A B Fig. 1. (A) Autophosphorylation activity of McsB. The autophosphorylation activity of McsB was evaluated by incubating 500 ng of each pro- tein with labelled ATP unless otherwise mentioned. The samples were resolved by 12% SDS ⁄ PAGE and stained with Coomassie Blue, and the phosphorylation activity was evaluated on a phosphorimager. The right panel shows a Coomassie Blue-stained gel, and the left panel shows the corresponding autoradiogram. Lane 1: McsB. Lane 2: McsA. Lane 3: McsB + McsA. Lane 4: McsB + McsA (1 lg). Lane 5: pro- tein marker. (B) Loss of activity of McsB mutants. The conserved tyrosine residues Tyr155 and Tyr210 were mutated in McsB to phenyala- nine, and kinase activity was measured by incubating 500 ng of protein with labelled ATP. The samples were separated by 12% SDS ⁄ PAGE, and phosphorylation activity was measured with a phosphorimager. Lane 1: McsB. Lane 2: McsB + McsA. Lane 3: McsBY155F. Lane 4: McsBY155F + McsA. Lane 5: McsBY210F. Lane 6: McsBY210F + McsA. A. R. Mattoo et al. McsB of Bacillus anthracis FEBS Journal 275 (2008) 6237–6247 ª 2008 The Authors Journal compilation ª 2008 FEBS 6239 ATP binding to McsB To investigate the enhanced autophosphorylation of McsB in the presence of McsA, binding of ATP to McsB was studied in the presence and absence of McsA. McsB of B. anthracis has two tryptophan residues (Trp14 and Trp148), whereas McsA has no tryptophan residue (Fig. S4). Trp148 is located in the ATP-guanidino phosphotransferase domain, which includes the important NEED motif and residues required for ATP and substrate binding [20,21]. The presence of Trp148 in the active site of McsB and the fact that McsA lacks tryptophan provided the tool with which to study ATP binding to McsB using fluo- rescence spectroscopy. Binding studies were carried out by measuring changes in the intrinsic tryptophan fluo- rescence of McsB upon addition of ATP (Fig. 2A). It was observed that addition of ATP significantly quenches fluorescence without changing the emission spectrum. The binding parameters of the experiment are represented in Table 1. Binding studies showed that 0.8 mol of ATP was bound per mol of McsB, with a binding affinity K a of 3.6 (± 0.4) · 10 3 m )1 . Studies on binding of ATP to McsB using the changes in intrinsic fluorescence were further carried out in the presence of McsA, which lacks a typtophan residue (Fig. 2B). The two proteins were preincubated at equi- molar concentrations for 30 min, and then titrated with similar concentrations of ATP as used for binding to McsB alone. Interestingly, the binding affinity of ATP for McsB increased by one order of magnitude, with K a of 2.5 (± 0.3) · 10 4 m )1 in the presence of McsA as compared to McsB alone (Fig. 2C). Earlier studies have shown that McsA of B. subtilis cannot bind ATP [5]. To determine whether McsA of B. anthracis also does not bind ATP, isothermal titra- tion calorimetry (ITC) experiments were performed. ITC allows the direct measurement of the equilibrium binding constant K a , the enthalpy of complex forma- tion (DH) and the complex stoichiometry of a protein– protein interaction without the need for modification of the proteins under investigation. The calorimetric titrations of McsA with Mg-ATP at 25 °C are shown in Fig. S5. The ITC experiments confirmed that McsA cannot bind ATP, and thus McsA has no direct role in the enhanced affinity of ATP for McsB. It is possible that the presence of McsA may induce a conforma- tional change in McsB upon interaction, leading to exposure of the residues required for optimum binding and hydrolysis of ATP. Earlier studies also showed that the phosphorylating capacity of Cap5B2, a tyro- sine kinase of Staphylococcus aureus, was expressed only in the presence of a stimulatory protein, either 2.0 x 10 7 A B C 1.5 x 10 7 1.0 x 10 7 5.0 x 10 6 1.2 x 10 6 9.0 x 10 5 6.0 x 10 5 3.0 x 10 5 Intensity (a.u.) Intensity (a.u.) 0.0 0.0 0.0 Wavelength (nm) 300 350 400 450 300 350 400 450 Wavelength (nm) –0.4 –0.2 ΔF-1 McsB 2.0 x 10 –4 0.0 4.0 x 10 –4 –0.8 –0.6 McsB + McsA ATP concentration (M) Fig. 2. Fluorescence experiments on Mg-ATP binding to McsB. Fluorescence spectra of (A) McsB (0.8 l M) and (B) an equimolar ratio (0.8 l M each) of McsB + McsA in the presence of increasing con- centrations of ATP (0.8–244 l M). All spectra were corrected by sub- traction of spectra obtained in buffer alone and buffer + Mg-ATP. The association constant K a for the McsB–Mg-ATP complex in the absence and presence of McsA was determined from the hyperbolic plots as shown in (C). DF)1 represents normalized fluorescence. McsB of Bacillus anthracis A. R. Mattoo et al. 6240 FEBS Journal 275 (2008) 6237–6247 ª 2008 The Authors Journal compilation ª 2008 FEBS Cap5A1 or Cap5A2, which enhances its affinity for the phosphoryl donor ATP [22,23]. There are several reports of eukaryotic kinases where binding of an interacting protein removes the inhibitory conforma- tion of the activation loop of the kinase, leading to its phosphorylation and further stabilizing the active form of the enzyme [24,25]. In order to estimate the kinetic parameters of ATP binding to McsB, surface plasmon resonance (SPR) measurements were carried out at different concentra- tions ranging from 6.25 lm to 100 lm Mg-ATP (Fig. 3). The binding affinity (K a ) of ATP for McsB was calculated to be 2.7 (± 0.3) · 10 3 m )1 . The low binding affinity, as also shown by fluorescence experi- ments, is in accordance with the binding of ATP to different kinases [26–28]. Binding of ATP to McsB was characterized by slow on-rates (k a ) and fast off-rates (k d ), as shown in Table 2. Studies of binding of McsB to its modulator McsA Complexes resulting from noncovalent protein–protein interactions play a fundamental role in most biological functions. McsB and its modulator McsA represent a unique model with which to study protein–protein interactions, where the presence of McsA enhances the autophosphorylation of McsB several-fold. We studied the binding pattern and the thermodynamic aspects of this interaction. The thermodynamics of the McsB– McsA interaction were analysed using ITC. Represen- tative calorimetric titrations of McsB with McsA at 25 °C are shown in Fig. 4. Each peak in the binding isotherms (Fig. 4, upper panel) represents a single injection of McsA. As observed from this experiment, the binding isotherm is characterized by strong heat changes that level off when the binding site on McsB becomes saturated. In the last injections of each titra- tion, only heat of dilution of McsA was observed. This was confirmed with parallel control experiments by injecting the same amount of McsA into the buffer (20 mm Hepes, pH 7.4, and 200 mm KCl). The values of heats of dilution were subtracted from the corre- sponding heat change associated with McsB–McsA interaction (Fig. 4, lower panel), in order to extract the thermodynamic parameters. The binding of McsA to McsB at 25 °C is characterized by a K a value of 5.0 (± 0.5) · 10 5 m )1 , DH=19.8 kcalÆmol )1 , and a Table 1. Binding parameters obtained from fluorescence spectro- scopic experiments performed in buffer (20 m M Hepes, pH 7.4, and 200 m M KCl]) at 25 °C. K a is the binding affinity value. The val- ues are means ± SE of three individual measurements. Sample K a (M )1 ) McsB–MgATP 3.6 (± 0.4) · 10 3 McsB–McsA 5.0 (± 0.5) · 10 5 (McsB–McsA)–MgATP 2.5 (± 0.3) · 10 4 300 400 300 200 Req 200 0 100 100 Response units (RU) 0.0 4.0 x 10 -5 8.0 x 10 -5 ATP concentration (M) 0 150 300 450 600 0 Time (s) Fig. 3. SPR experiments on Mg-ATP binding to McsB. Representative sensorgrams for ATP binding are presented in the left panel. The con- centrations of ATP (prepared in 20 m M Hepes buffer, 150 mM NaCl and 5 mM MgCl 2 ) used were 6.25, 12.5, 25, 50 and 100 lM from the bottom up. The lines are best fits to the steady-state RU values, which are directly proportional to the analyte concentration (C). The right panel shows a direct binding plot of R eq versus concentration of ATP. The lines are obtained by nonlinear least-squares fits of the data. Table 2. Kinetic parameters of Mg-ATP–McsB and McsA–McsB interactions obtained from SPR experiments performed in buffer (20 m M Hepes, pH 7.4, 150 mM NaCl, 50 lM EDTA and 0.005% surfactant P20) at 25 °C. k a is the association rate constant, and k d is the dissociation rate constant. K a is the binding affinity value obtained for the McsB–Mg-ATP and McsA–McsB interactions. The values are means ± SE of three individual measurements. Sample k a (M )1 Æs )1 ) k d (s )1 ) K a (M )1 ) McsB–Mg-ATP 1.7 · 10 1 6.2 · 10 )3 2.7 (± 0.3) · 10 3 McsB–McsA 7.5 · 10 1 3.8 · 10 )4 2.0 (± 0.3) · 10 5 A. R. Mattoo et al. McsB of Bacillus anthracis FEBS Journal 275 (2008) 6237–6247 ª 2008 The Authors Journal compilation ª 2008 FEBS 6241 stoichiometry of 0.8 ($ 1) (Table 3). A complete ther- modynamic description of the binding, including the free energy of binding (DG) and the change in entropy (DS), was calculated using DG=)RT ln(K a ) = DH– TDS, where R is the gas constant and T is the temper- ature in kelvin. The complete thermodynamic and binding parameters are given in Table 3. The associa- tion of McsA with McsB is endothermic, and thus enthalphically unfavourable (DH > 0). The interaction between the two proteins was observed regardless of the unfavourable large positive DH value associated with the interaction. This observation indicated that the interaction was spontaneous (DG < 0), thus requiring a large positive change in entropy. The over- all enthalpy change may be due to conformational enthalpy, interaction enthalpy or solvation (hydra- tion ⁄ dehydration) enthalpy that arise due to the removal or intake of water molecules at the interface. The conformational enthalpy is generally considered to be exothermic, as formation of secondary structure is favourable. Similarly, interaction enthalpy is also exo- thermic, as it involves the formation of new noncova- lent interactions such as electrostatic attraction, van der Waals interactions, and hydrogen bonds [29]. However, as the overall enthalpy change is endother- mic, these two terms may be negligible, and the endo- thermic enthalpy of association may be contributed by large positive solvation enthalpy that arises due to the release of ordered water molecules from the McsB– McsA interface (i.e. dehydration). Thermodynamic data from various sources, such as the nonpolar phase to water, protein folding and ligand binding to protein through hydrophobic effects, are accompanied by bur- ial of the nonpolar surface from water [30–32]. It has been suggested that the hydrophobic surfaces induce orientation in the water–water hydrogen bonds in the first hydration shell and that this ordered water is released on burial of the surface [31,32]. Protein– protein complex formation is commonly thermody- namically unfavourable in terms of enthalpy; however, positive changes in entropy, primarily due to dehydra- tion of the protein interfaces, provide thermodynamic stability for the complex and drive the interaction [33,34]. In order to estimate the kinetic parameters of the binding of McsA to McsB, the SPR measurements were carried out at various concentrations of McsA as discussed in Experimental procedures (Fig. 5A). The resulting kinetic constants (Table 2) revealed that the interaction between McsA and McsB takes place with low association (k a = 7.5 · 10 1 m )1 Æs )1 ) and fast dis- sociation (k d = 3.8 · 10 )4 s )1 ) rates. The equilibrium binding constant K a for the McsB–McsA interaction was found to be 2.0 (± 0.3) · 10 5 m )1 , which is in accordance with the K a obtained from the ITC experi- ments (Table 3). In most cases, the interaction between Fig. 4. Binding of tyrosine kinase McsA to its modulator McsB. Both McsA and McsB were dialysed against the same buffer (20 m M Hepes, pH 7.4, and 200 mM KCl), and the titrations were performed in the same buffer. McsA (833 l M) was titrated into McsB (25 l M). Data analysis was performed with ORIGIN 7.0 soft- ware, provided by MicroCal. The data were fitted to a model for a single class of binding sites (solid line). Table 3. Thermodynamic parameters of the McsA–McsB interaction obtained from calorimetric experiments performed in buffer (20 mM Hepes, pH 7.4, and 200 mM KCl) at 25 °C. The values are obtained by fitting the ITC titration data by applying the single-site model. DH is binding enthalpy change, DS is binding entropy change. and DG 25 °C is the free energy of the McsA–McsB interaction at 25 °C obtained using DG = )RT lnK a . K a is the binding affinity value obtained for the McsA–McsB interaction from both spectroscopic and calorimetric experiments. The values are means ± SE of three individual measurements. Ligand DH (kcalÆmol )1 ) DS (calÆmol )1 ÆK )1 ) DG 25 °C (kcalÆmol )1 ) K a (M )1 ) McsA 19.8 ± 2.0 26.2 ± 2.7 )7.8 ± 0.8 5.0 (± 0.5) · 10 5 McsB of Bacillus anthracis A. R. Mattoo et al. 6242 FEBS Journal 275 (2008) 6237–6247 ª 2008 The Authors Journal compilation ª 2008 FEBS a protein kinase and its substrate is transient and of low affinity [35,36]. The moderate to low value of the binding affinity and kinetic parameters suggests that binding of the modulator (McsA) to the tyrosine kinase (McsB) follows the same principle as substrate binding to a kinase. It seems that this moderate inter- action is sufficient to induce a conformational change in McsB that enhances the binding of ATP to this tyrosine kinase, as discussed above. Furthermore, the presence of tryptophan residues in McsB and their absence in McsA (as discussed above) can also be used to study the interaction between the proteins by measuring changes in the intrinsic trypto- phan fluorescence of McsB by increasing the con- centration of McsA. Titration of McsB by McsA decreased the fluorescence intensity of tryptophan sig- nificantly before reaching saturation (Fig. 5B). The decrease in the intrinsic fluorescence of McsB at 340 nm was monitored in the presence of increasing concentrations of McsA, which allowed determination of the K a value of 5.0 (± 0.5) · 10 5 m )1 and the stochiometry of 0.9, very similar to those obtained from ITC and SPR studies. Conclusion In this study, we show that B. anthracis lacks some of the important tyrosine kinases that are active in the closely related nonpathogenic B. subtilis. This study adds to the growing list of nonfunctional or absent genes in B. anthracis in comparison to other species of the genus Bacillus, which can be attributed to the path- ogenicity of this organism. It has been hypothesized that specialization of B. anthracis as a pathogen could have reduced the range of environmental stimuli to which it is exposed. This, along with the presence of the pathogenic plasmids pX01 and pX02, may have rendered some of its tyrosine kinases redundant, ulti- mately resulting in the loss of ptkA and ptkB genes. Our data provide an insight into the enhanced activity of the tyrosine kinase, McsB, in the presence of the modulator McsA. The moderate binding of McsA to McsB, which is entropically driven, appears to induce a conformational change in McsB resulting in the increased ATP binding. Recently, a tyrosine kinase from S. aureus, Cap5B2, has also been shown to require the presence of modulators Cap5A1 ⁄ Cap5A2 for ATP binding and utilization. In the absence of activator proteins, this kinase is completely inactive. However, both McsB and Cap5B2 have completely dif- ferent modulators that have no similarity at the sequence level. Cap5B2 is an ATPase-type tyrosine kinase with Walker A and Walker B domains, unlike McsB, which has a guanidino-phosphotransferase domain. Moreover, McsA is phosphorylated by McsB, which is not the case with Cap5A1 or Cap5A2. Recent –0.2 0.0 –0.6 –0.4 ΔF-1 0.0 4.0 x 10 -5 2.0 x 10 -5 –0.8 1.2 x 10 7 8.0 x 10 6 4.0 x 10 6 0.0 Intensity (a.u.) 300 350 400 450 Wavelength (nm) 300 A B 200 100 Response units (RU) 0 200 400 600 0 Time (s) 300 200 Req 0 100 0.0 4.0 x 10 -6 8.0 x 10 -6 McsA concentration (M) McsA concentration ( M) Fig. 5. (A) Kinetic measurements of McsA– McsB interaction. Sensograms of the bind- ing of increasing concentrations of McsA to McsB (immobilized on an Ni 2+ –nitrilotroace- tic acid chip). The concentrations of McsA used were 0.625, 1.25, 2.5, 5 and 10 l M from the bottom up (left panel). Data points represent the equilibrium average response. The solid line (right panel) represents the theoretical curve that was globally calculated by nonlinear least-squares fits of the data provided by BIAEVALUATION 3.1 software (Bia- core). (B) Titration curve of McsB with McsA. The titration of McsB (0.8 l M) was performed with increasing concentrations of McsA (0.2–45 l M). The binding constant K a for McsA binding to McsB was determined from the hyperbolic plot as shown in the right panel. DF)1 represents normalized fluorescence. A. R. Mattoo et al. McsB of Bacillus anthracis FEBS Journal 275 (2008) 6237–6247 ª 2008 The Authors Journal compilation ª 2008 FEBS 6243 structural studies have tried to address the molecular basis for the regulatory mechanism of the Cap5A1– Cap5B2 complex, and have given insights into their copolymerase function. Similar crystallization studies are required for the McsB–McsA complex, to unravel the molecular details of enhanced phosphorylation. In conclusion, we suggest that all prokaryotic tyrosine kinases with kinase and modulator domains on different polypeptides may utilize a similar molecular mechanism for triggering protein-tyrosine kinase activity. Experimental procedures Materials The genomic DNA isolated from B. anthracis Sterne strain was used for cloning BAS0079 and BAS0080. E. coli strains DH5-a and BL21-kDE3 were used for gene manipulation and protein expression, respectively. Biochemical reagents were purchased from Sigma-Aldrich (St Louis, MO, USA), Merck (Darmstadt, Germany) and Bangalore Genei India Ltd (Bangalore, India). Bacterial culture media were purchased from HiMedia laboratories (Mumbai, India). Ni 2+ –nitrilotriacetic acid resin for affinity purification was purchased from Qiagen (Hilden, Germany). Sensor chip Ni 2+ –nitrilotriacetic acid was obtained from Biacore AB (Uppsala, Sweden). DNA-modifying enzymes were obtained from Roche (Basel, Switzerland). [ 32 P]ATP[cP] was purchased from BRIT (Hyderabad, India). Plasmid construction and mutagenesis The cloning of mcsA and mcsB and the mutagenic analysis was performed as previously described [37]. The genes were cloned in the pROEX-HTc plasmid. The vector pROEX- HTc has sequences coding for six histidine residues at the N-terminus. All of the experiments were performed with McsB and McsA containing six histidine residues at the N-terminus unless otherwise mentioned. The details of primers used in the study are given in Table S1. Purification of McsB, McsA and mutant proteins The purification of McsB, McsA and mutant proteins was performed as previously described [37], with certain modifi- cations. When D 600 nm of the E. coli BL21-kDE3 (trans- formed with plasmids containing McsA, McsB and its mutants) culture reached 0.6, isopropyl thio-b-d-galactoside was added to a final concentration of 0.4 mm, and induc- tion was performed at 18 °C for 8 h. The protein was dialy- sed against the buffer (20 mm Hepes, pH 7.4, 200 mm KCl) to remove immidazole, before being used for the biochemi- cal and biophysical assays. Autophosphorylation of McsB and its mutants Autophosphorylation activity of the purified McsB and mutant proteins was checked as previously described [37]. In brief, 500 ng of the purified McsB and the same amount of McsA was incubated with 10 lCi of [ 32 P]ATP[cP] in a final reaction volume of 20 lL prepared with HMD buffer (20 mm Hepes pH 7.4, 5 mm MgCl 2 ,1mm dithiothreitol). The reaction was allowed to continue for 30 min, and terminated by addition of 2 lLof5· SDS sample buffer. The samples were boiled for 5 min and separated by 12% SDS ⁄ PAGE. The gel was fixed in 40% methanol, dried, and evaluated in an FLA 2000 (Fujifilm) phosphorimager after exposure for 30 min. SPR experiments The SPR studies were carried out as described earlier [38,39]. In brief, nitrilotriacetic acid chips were used to bind histidine-tagged McsB. The SPR experiments were per- formed at 25 °C in filtered, degassed 20 mm Hepes buffer (pH 7.4) containing 150 mm NaCl, 50 lm EDTA and 0.005% surfactant P20. Protein ⁄ ATP solutions were pre- pared by serial dilution from the stock solution and injected from 7 mm plastic vials with pierceable plastic crimp caps. Protein ⁄ ATP solution flow was continued until a constant steady-state response was obtained. Protein ⁄ ATP flow was then replaced by buffer flow to monitor dissociation of the complex. The reference response from the blank cell was subtracted from the response in the immobilized protein cell to give a signal (RU, response units) that is directly proportional to the amount of bound ATP ⁄ protein. Sensor- grams, RU versus time, at different concentrations for binding of MgATP ⁄ McsA to McsB were obtained, and the RU in the steady-state region were determined by linear averaging over a selected time span. The data obtained from the SPR experiments was analysed using the equation R eq ¼ RU max  K a  C=ð1 þ K a  CÞ where RU max is the maximum response per bound protein or ATP, K a is the macroscopic binding constant, C is the analyte concentration (m), and R eq is the steady-state bind- ing level. ITC experiments ITC experiments were performed using a MicroCal VP-ITC-type microcalorimeter (MicroCal Inc.) at 25 °C [26–29]. Temperature equilibration prior to experiments was allowed for 1–2 h. All solutions were thoroughly degassed before use by stirring under vacuum. Protein samples (McsA and McsB) were prepared in the same dialysis buffer (20 mm Hepes, pH 7.4, 200 mm KCl). A typical titration experiment consisted of consecutive injections of 5 lL of the McsB of Bacillus anthracis A. R. Mattoo et al. 6244 FEBS Journal 275 (2008) 6237–6247 ª 2008 The Authors Journal compilation ª 2008 FEBS titrating ligand (in 25 steps, at 5 min intervals, into the pro- tein solution in the cell with a volume of 2 mL). The titra- tion data were corrected for the small heat changes observed in the control titrations of ligands into the buffer. Data analysis was performed with origin 7.0 software, provided by MicroCal, using equations and curve-fitting analysis to obtain least-square estimates of the binding enthalpy, stoichiometry, and binding constant. Binding stoichiometries were derived on the assumption that the two proteins were fully active with respect to binding. Fluorescence measurements Binding of the nucleotide Mg-ATP to McsB and an equi- molar ratio of McsB ⁄ McsA was monitored by changes in the intrinsic tryptophan fluorescence of McsB. The experi- ments were performed as described earlier, at 25 °C using a Fluoromax 4 spectrofluorimeter [26,40,41]. The excitation wavelength was 290 nm (slit width 5 nm), and emission was observed between 300 and 450 nm (slit width 5 nm). McsB protein was diluted to 0.8 lm in buffer containing 20 mm Hepes (pH 7.4) and 100 mm KCl, titrated with increasing concentrations of McsA ⁄ Mg-ATP. All spectra were cor- rected for buffer fluorescence, inner filter effects of ATP, and dilution (never exceeding 2% of the original volume). The binding constant (K a ) for Mg-ATP or McsA binding to McsB was determined by fitting of a hyperbolic plot to the titration data. Acknowledgements Financial support to Abid R. Mattoo from the Coun- cil of Scientific and Industrial Research, India and to Amit Arora from the University Grants Commission, India is acknowledged. The project was supported by CSIR Task Force Project NWP-0038. We would like to thank Dr V. C. Kalia for helpful suggestions. References 1 Gay YA, Jamil S & Drews SJ (1999) Expression and characterization of the Mycobacterium tuberculosis ser- ine ⁄ threonine protein kinase PknB. Infect Immun 67, 5676–5682. 2 Madec E, Laszkiewicz A, Iwanicki A, Obuchowski M & Simone S (2002) Characterization of a membrane- linked Ser ⁄ Thr protein kinase in Bacillus subtilis impli- cated in developmental processes. Mol Microbiol 2, 571–586. 3 Cozzone AJ, Grangeasse C, Doublet P & Duclos B (2004) Protein phosphorylation on tyrosine in bacteria. Arch Microbiol 181, 171–181. 4 Grangeasse C, Cozzone AJ, Deutscher J & Mijakovic I (2006) Tyrosine phosphorylation: an emerging regula- tory device of bacterial physiology. Trends Biochem Sci 32, 86–94. 5 Kirstein J, Zu ¨ hlke D, Gerth U, Turgay K & Hecker M (2005) A tyrosine kinase and its activator control the activity of the CtsR heat shock repressor in B. subtilis. EMBO J 24, 3435–3445. 6 Cozzone AJ (2005) Role of protein phosphorylation on serine ⁄ threonine and tyrosine in the virulence of bacte- rial pathogens. J Mol Microbiol Biotechnol 9, 198–213. 7 Klein G, Dartigalongue C & Raina S (2003) Phosphor- ylation-mediated regulation of heat shock response in Escherichia coli. Mol Microbiol 48, 269–285. 8 Grangeasse C, Obadia B, Mijakovic I, Deutscher J, Cozzone AJ & Double P (2003) Autophosphorylation of the Escherichia coli protein kinase Wzc regulates tyrosine phosphorylation of Ugd, a UDP-glucose dehydrogenase. J Biol Chem 278, 39323–39329. 9 Mijakovic I, Poncet S, Boe ¨ l G, Maze ´ A, Gillet S, Jamet E, Decottignies P, Grangeasse C, Doublet P, Mare ´ chal PL et al. (2003) Transmembrane modulator dependent bacterial tyrosine kinase activates UDP-glucose dehy- drogenases. EMBO J 22, 4709–4718. 10 Mijakovic I, Petranovic D, Macek B, Cepo T, Mann M, Davies J, Jensen PR & Vujaklija D (2006) Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine. Nucleic Acids Res 20, 1588–1596. 11 Brunsing RL, Chandra LC, Tang S, Christina C, Han- cock LE, Perego M & Hoch JA (2005) Characterization of sporulation histidine kinases of Bacillus anthracis. J Bacteriol 187, 6972–6981. 12 de Been M, Francke C, Moezelaar R, Abee T & Siezen RJ (2006) Comparative analysis of two-component sig- nal transduction systems of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis . Microbiology 152, 3035–3048. 13 Mijakovic I, Petranovic D, Bottini N, Deutscher J & Jensen PR (2005) Protein-tyrosine phosphorylation in Bacillus subtilis. J Mol Microbiol Biotechnol 9, 189– 197. 14 Ogura M, Matsuzawa A, Yoshikawa H & Tanaka T (2004) Bacillus subtilis SalA (YbaL) negatively regulates expression of scoC, which encodes the repressor for the alkaline exoprotease gene, aprE. J Bacteriol 186, 3056– 3064. 15 Petranovic D, Michelsen O, Zahradka K, Silva C, Petranovic M, Jensen PR & Mijakovic I (2007) Bacillus subtilis strain deficient for the protein-tyrosine kinase PtkA exhibits impaired DNA replication. Mol Microbiol 63, 1797–1805. 16 Quisel JD & Grossman AD (2000) Control of sporula- tion gene expression in Bacillus subtlis by the chromo- some partitioning protein Soj (ParA) and Spo0J (ParB). J Bacteriol 182, 3446–3451. A. R. Mattoo et al. McsB of Bacillus anthracis FEBS Journal 275 (2008) 6237–6247 ª 2008 The Authors Journal compilation ª 2008 FEBS 6245 17 Levin PA, Shim JJ & Grossman AD (1998) Effect of minCD on FtsZ ring position and polar septation in Bacillus subtilis. J Bacteriol 180, 6048–6051. 18 Koonin EV (2005) Orthologs, paralogs, and evolution- ary genomics. Annu Rev Genet 39, 309–338. 19 Narayan A, Sachdeva P, Sharma K, Saini AK, Tyagi AK & Singh Y (2007) Serine threonine protein kinases of mycobacterial genus: phylogeny to function. Physiol Genomics 29, 66–75. 20 Yousef MS, Fabiola F, Gattis JL, Somasundaram T & Chapman MS (2002) Refinement of the arginine kinase transition-state analogue complex at 1.2 A resolution, mechanistic insights. Acta Crystallogr D Biol Crystallogr 58, 2009–2017. 21 Gattis JL, Ruben E, Fenley MO, Ellington WR & Chapman MS (2004) The active site cysteine of arginine kinase, structural and functional analysis of partially active mutants. Biochemistry 43, 8680–8689. 22 Soulat D, Jault JM, Duclos B, Geourjon C, Cozzone AJ & Grangeasse C (2006) Staphylococcus aureus oper- ates protein-tyrosine phosphorylation through a specific mechanism. J Biol Chem 281, 14048–14056. 23 Olivares-Illana V, Meyer P, Bechet E, Gueguen-Chai- gnon V, Soulat D, Lazereg-Riquier S, Mijakovic I, Deutscher J, Cozzone AJ, Lapre ´ vote O et al. (2008) Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus. PLoS Biol 6, 1321–1331. 24 Jeffrey PD, Russo AA, Polyak K, Gibbs E, Hurwitz J, Massague J & Pavletich NP (1995) Mechanism of CDK activation revealed by the structure of a cyclinA–CDK2 complex. Nature 376, 313–320. 25 Pellicena P & Kuriyan J (2006) Protein–protein inter- actions in the allosteric regulation of protein kinases. Curr Opin Struct Biol 16 , 702–709. 26 Mattoo AR, Zaman MS, Dubey GP, Arora A, Narayan A, Jailkhani N, Rathore K, Maiti S & Singh Y (2008) Spo0B of Bacillus anthracis – a protein with pleiotropic functions. FEBS J 275, 739–752. 27 Forstner M, Berger C & Wallimann T (1999) Nucleotide binding to creatine kinase: an isothermal titration microcalorimetry study. FEBS Lett 461, 111–114. 28 Flachner B, Kovari Z, Varga A, Gugolya Z, Vonderviszt F, Szabo GN & Vas M (2004) Role of phosphate chain mobility of MgATP in completing the 3-phosphoglycerate kinase catalytic site: binding, kinetic, and crystallographic studies with ATP and MgATP. Biochemistry 43, 3436–3449. 29 Abraham T, Lewis RN, Hodges RS & McElhaney RN (2005) Isothermal titration calorimetry studies of the binding of a rationally designed analogue of the anti- microbial peptide gramicidin S to phospholipid bilayer membranes. Biochemistry 44, 2103–2112. 30 Blokzijl W & Engberts JBFN (1993) Hydrophobic effects – opinion and facts. Angew Chem Intl Ed Engl 32, 1545–1579. 31 Xu H & Dill KA (2005) Water’s hydrogen bond in the hydrophobic effect: a simple model. J Phys Chem B 109, 23611–23617. 32 Sharp KA & Madan B (1997) Hydrophobic effect, water structure, and heat capacity changes. J Phys Chem B 101, 4343–4348. 33 Kresheck GC, Vitello LB & Erman JE (1995) Calori- metric studies on the interaction of horse ferricyto- chrome c and yeast cytochrome c peroxidase. Biochemistry 34, 8398–8405. 34 Jelesarov I & Bosshard HR (1994) Thermodynamics of ferredoxin binding to ferredoxin, NADP + reductase and the role of water at the complex interface. Bio- chemistry 33, 13321–13328. 35 Maly DJ, Allen JA & Shokat KM (2004) A mecha- nism-based cross-linker for the identification of kinase–substrate pairs. J Am Chem Soc 126, 9160– 9161. 36 Manning BD & Cantley LC (2002) Hitting the target: emerging technologies in the search for kinase sub- strates. Sci STKE 162, 1–4. 37 Chopra P, Singh A, Koul A, Ramachandran S, Drlica K, Tyagi AK & Singh Y (2003) Cytotoxic activ- ity of nucleoside diphosphate kinase secreted from Mycobacterium tuberculosis. Eur J Biochem 270, 625– 634. 38 Nieba L, Nieba-Axmann SE, Persson A, Ha ¨ ma ¨ la ¨ inen M, Edebratt F, Hansson A, Lidholm J, Magnusson K, Karlsson AF & Plu ¨ ckthun A (1997) BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Anal Biochem 252, 217–228. 39 Jisa E & Jungbauer A (2003) Kinetic analysis of estro- gen receptor homo- and heterodimerization in vitro. J Steroid Biochem Mol Biol 84, 141–148. 40 Kunrong C & Koland JG (1998) Nucleotide-binding properties of kinase deficient epidermal-growth-factor receptor mutants. Biochem J 330, 353–359. 41 Ramaen O, Masscheleyn S, Duffieux F, Pamlard O, Oberkampf M, Lallemand JY, Stoven V & Jacquet E (2003) Biochemical characterization and NMR studies of the nucleotide-binding domain 1 of multi- drug-resistance-associated protein 1: evidence for interaction between ATP and Trp653. Biochem J 376, 749–756. Supporting information The following supplementary material is available: Fig. S1. (A) Comparison of the yveL ( ptkB ) gene locus of Bacillus subtilis with that of Bacillus anthracis. (B) McsB of Bacillus anthracis A. R. Mattoo et al. 6246 FEBS Journal 275 (2008) 6237–6247 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... used for cloning of McsB and McsA, and for creating site-specific mutants of Tyr155 and Tyr210 of McsB McsB of Bacillus anthracis This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed.. .A R Mattoo et al Multiple sequence alignment of the McsB from different species of prokaryotes Fig S2 Acid–base and heat stability of phosphorylated McsB in the presence of McsA Fig S3 CD spectra of McsB and its mutants Fig S4 McsB has two tryptophan residues (Trp14 and Trp148), whereas McsA has none Fig S5 Binding of ATP to McsA using the ITC method Table S1 List of primers used for cloning of. .. content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 6237–6247 ª 2008 The Authors Journal compilation ª 2008 FEBS 6247 . Identification, characterization and activation mechanism of a tyrosine kinase of Bacillus anthracis Abid R. Mattoo, Amit Arora, Souvik Maiti and Yogendra. McsB of B. anthracis is a tyro- sine kinase and that Tyr155 and Tyr210 are the sites of autophosphorylation. A B Fig. 1. (A) Autophosphorylation activity of