The presence of McsA not only enhanced the autophosphorylation activity of McsB, but also resulted in phosphorylation of McsA.. anthracis was enhanced in the presence of an activator pro
Trang 1of 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 103 to 104 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 (ka) and fast dissociation rates (kd) 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.
Trang 2and 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 anthracisis 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 tyrotyro-sine 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
Trang 3B 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-aciswas 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
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 (95C) 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.
Trang 4ATP 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 Ka of 3.6 (± 0.4)· 103m)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 Ka of 2.5 (± 0.3)· 104m)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 Ka, 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 25C 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
0.0
0.0
0.0
Wavelength (nm)
Wavelength (nm)
–0.4 –0.2
McsB
2.0 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 Kafor 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.
Trang 5Cap5A1 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 activaconforma-tion 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 (Ka) of ATP for McsB
was calculated to be 2.7 (± 0.3)· 103m)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 (ka) and fast off-rates
(kd), 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
25C 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 25C is characterized by a Ka value of 5.0 (± 0.5)· 105m)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 Ka is the binding affinity value The
val-ues are means ± SE of three individual measurements.
300
400 300
200
200
0
100 100
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 m M NaCl and 5 m M MgCl2) used were 6.25, 12.5, 25, 50 and 100 l M 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 Reqversus 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 m M NaCl, 50 l M EDTA and 0.005% surfactant P20) at 25 C ka is the association rate constant, and kd
is the dissociation rate constant Ka 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 ka( M )1Æs)1) k
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
Trang 6stoichiometry 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(Ka) =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 (ka= 7.5· 101m)1Æs)1) and fast dis-sociation (kd= 3.8· 10)4s)1) rates The equilibrium binding constant Ka for the McsB–McsA interaction was found to be 2.0 (± 0.3)· 105m)1, which is in accordance with the Ka 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 m M 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 m M Hepes, pH 7.4, and 200 m M 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 DG25 C is the free energy of the McsA–McsB interaction at 25 C obtained using DG = )RT lnKa Kais 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)1ÆK)1) DG25 C (kcalÆmol)1) Ka ( M )1)
Trang 7a 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 Ka value of 5.0 (± 0.5)· 105m)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
0.0 2.0 x 10 -5 4.0 x 10 -5 –0.8
1.2 x 10 7
8.0 x 10 6
4.0 x 10 6
0.0
300 350 400 450 Wavelength (nm)
300
A
B
200
100
0 200 400 600 0
Time (s)
300
200
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 increasbind-ing concentrations of McsA to
McsB (immobilized on an Ni2+
–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 Ka
for McsA binding to McsB was determined
from the hyperbolic plot as shown in the
right panel DF)1 represents normalized
fluorescence.
Trang 8structural 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)
purchased from Qiagen (Hilden, Germany) Sensor chip
AB (Uppsala, Sweden) DNA-modifying enzymes were
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 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
(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
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 biophysibiochemi-cal 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
final reaction volume of 20 lL prepared with HMD buffer (20 mm Hepes pH 7.4, 5 mm MgCl2, 1 mm dithiothreitol) The reaction was allowed to continue for 30 min, and terminated by addition of 2 lL of 5· SDS sample buffer The samples were boiled for 5 min and separated by 12%
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
(pH 7.4) containing 150 mm NaCl, 50 lm EDTA and
pre-pared by serial dilution from the stock solution and injected from 7 mm plastic vials with pierceable plastic crimp caps
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
Sensor-grams, RU versus time, at different concentrations for
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
bind-ing level
ITC experiments
[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
Trang 9titrating 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
the intrinsic tryptophan fluorescence of McsB The
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
cor-rected for buffer fluorescence, inner filter effects of ATP,
and dilution (never exceeding 2% of the original volume)
The binding constant (Ka) 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
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
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
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
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
Trang 1017 Levin PA, Shim JJ & Grossman AD (1998) Effect of
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 cytoferricyto-chrome c peroxidase
Biochemistry 34, 8398–8405
34 Jelesarov I & Bosshard HR (1994) Thermodynamics of
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)