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: ;
(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.
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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
Multiple sequence alignment of the McsB from differ-
ent species of prokaryotes.
Fig. S2. Acid–base and heat stability of phosphory-
lated 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 McsB
and McsA, and for creating site-specific mutants of
Tyr155 and Tyr210 of McsB.

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A. R. Mattoo et al. McsB of Bacillus anthracis
FEBS Journal 275 (2008) 6237–6247 ª 2008 The Authors Journal compilation ª 2008 FEBS 6247