From the Unité de Biochimie Structurale, URA 2185 CNRS, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris, cedex 15, France
Received for publication, January 21, 2003
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ABSTRACT |
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With the advent of the sequencing programs of
prokaryotic genomes, many examples of the presence of serine/threonine
protein kinases in these organisms have been identified. Moreover,
these kinases could be classified as homologues of those belonging to the well characterized superfamily of the eukaryotic serine/threonine and tyrosine kinases. Eleven such kinases were recognized in the genome
of Mycobacterium tuberculosis. Here we report the crystal structure of an active form of PknB, one of the four M. tuberculosis kinases that are conserved in the downsized genome
of Mycobacterium leprae and are therefore presumed to play
an important role in the processes that regulate the complex life cycle
of mycobacteria. Our structure confirms again the extraordinary
conservation of the protein kinase fold and constitutes a landmark that
extends this conservation across the evolutionary distance between high eukaryotes and eubacteria. The structure of PknB, in complex with a
nucleotide triphosphate analog, reveals an enzyme in the active state
with an unprecedented arrangement of the Gly-rich loop
associated with a new conformation of the nucleotide The reversible phosphorylation of proteins constitutes one of the
most widespread mechanisms involved in the regulation of different
processes in living organisms. It was first suggested in 1955 as a way
of modulating the enzymatic activity of the glycogen phosphorylase
enzyme (1). Since this discovery, two main superfamilies of protein
kinases were identified, one including
STPKs1 and PTKs (2) and that
of His kinases (3). For a long time, the former were only found in
eukaryotes, and the latter were only found in prokaryotes. In this
paradigm, proteins from each superfamily were supposed to play
analogous roles in the essentially different organization of signal
transduction in both phyla. However, in 1978 the phosphorylation of
serine/threonine residues in bacteria was demonstrated (4). Since then
several bacterial genes encoding STPKs, PTKs, or their counterpart
phosphatases have been identified and cloned (for a review, see Ref.
5). Their existence raises the question of their role in signal
transduction pathways, possibly involving complex networks equivalent
to those widely studied in eukaryotes (6). Interestingly morphological
richness and complicated biochemical differentiation processes, often
directed by environmental changes, seem to be common themes in the life cycle of the bacteria possessing more than three genes encoding STPKs
(7).
The genome of Mycobacterium tuberculosis has 11 genes
encoding putative STPKs (8). Indeed the products of some of these genes
have been confirmed to possess kinase activity
(9-12).2 Only four of these
genes, namely pknA, pknB, pknG, and
pknL, appear to be conserved in the downsized genome of the
close relative pathogen Mycobacterium leprae (13) and hence
may be supposed to play fundamental roles in the biology of
mycobacteria. Notably, the pknA and pknB genes
are found next to each other in a genomic region that is conserved in
M. leprae and also in other Actinomycetales like
Streptomyces coelicolor. This region also includes one gene encoding a putative serine/threonine phosphatase and genes for proteins
involved in the cell wall synthesis. These characteristics strongly
suggest that this region could actually constitute a functional operon.
On the other hand, it is worth noting that the number of two-component
systems identified in the M. tuberculosis genome, probably no more than 15 (8), is roughly one-third of that found, for example, in Escherichia coli, a bacterium with a similar genome size.
What may be the role of these STPKs and phosphatases in the adaptation of mycobacteria to the intricate interactions that they maintain with
their hosts remains an open question. It would also be of great
interest to assess whether the two-component systems and the
serine/threonine kinases and phosphatases may be interconnected in the
regulation of certain functions.
PknB is a member of the recently described (14) Pkn2 subfamily of STPKs
found in prokaryotes. This subfamily shows the highest sequence
similarity with the eukaryotic orthologues, and it might have appeared
from early horizontal transfer from eukaryotes to bacteria (14). PknB
is predicted to consist of 626 amino acids with a transmembrane segment
dividing the protein into an N-terminal intracellular domain and a
C-terminal extracellular domain. The N-terminal domain of PknB includes
a kinase domain and juxtamembrane linker of 52 residues. The PknB
kinase core retains all the essential amino acids and sequence
subdomains (Fig. 1A) that are required for a functional STPK
(2) despite a modest degree of identity when compared with eukaryotic
STPKs (Table I). Actually the full-length PknB protein has been characterized as an active serine/threonine kinase (9).
-phosphoryl
group. It presents as well a partially disordered activation loop,
suggesting an induced fit mode of binding for the so far unknown
substrates of this kinase or for some modulating factor(s).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Sequence identity and structural similarity between PknB and some
eukaryotic kinases
This contribution presents the crystal structure of the recombinant
kinase domain of M. tuberculosis PknB in complex with an ATP
analog, AMP-PCP, and constitutes the first reported structure of a
bacterial STPK. The structure reveals a number of features that explain
the intrinsic active state of this construction and suggests an induced
fit mode of binding for either the hitherto unknown PknB substrates or
other external modulating factor(s).
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EXPERIMENTAL PROCEDURES |
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Protein Expression and Purification--
The N-terminal sequence
of the pknB gene (Rv0014c) encoding the cytoplasmic domain
of the PknB protein (residues 1-331) was initially cloned from the
MTCY10H4 cosmid. The digested and purified PCR product was ligated into
the T7 RNA polymerase-based pET28 expression vector (Novagen) providing
an N-terminal tag of 20 amino acids with a His6 motif.
Expression of this construction in E. coli resulted in a
highly heterogeneous protein, possibly as a result of variable
autophosphorylation events in the juxtamembrane linker (data not
shown). Multiple sequence alignment with other members of the protein
superfamily and comparative modeling suggested Gly-279 as a probable C
terminus of the last -helix (
I) of the PknB kinase domain. Hence
a shorter construct of this domain (residues 1-279) was obtained by
the introduction of a stop codon through site-directed mutagenesis. The
PknB-(1-279) protein was expressed in E. coli
BL21(DE3). Bacteria were grown at 37 °C until late log phase in LB
medium supplemented with kanamycin (30 µg/ml) and induced for 12-16
h at 15 °C by addition of 1 mM
isopropyl-1-thio-
-D-galactopyranoside. The
bacterial pellet was resuspended and sonified in 50 mM
Hepes buffer, pH 7, 0.2 M NaCl including protease
inhibitors. The lysate was cleared by centrifugation (20,000 × g for 1 h). The supernatant containing soluble proteins
was applied to Ni2+-loaded HiTrap chelating columns
(Amersham Biosciences) using an fast protein liquid
chromatography system and eluted through an imidazole gradient
(0-0.5 M). A further step of gel filtration in a Superdex
75 column was performed to separate the monomeric protein from
aggregated material. The protein was subsequently concentrated by means
of Macro- and Microsep concentrators (Pall/Gellman) up to a
concentration of ~10 mg/ml. Protein concentration was determined
using the Bio-Rad protein assay. Purity of the samples was checked by
SDS-PAGE.
Protein Kinase and Inhibition Assays--
One unit of the enzyme
control PKA (New England Biolabs) or 1 µM purified
PknB was incubated with 10 µM myelin basic protein (Invitrogen) as model substrate. The assays were carried out in 20 µl
of PKA buffer (50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2) or PknB buffer (50 mM Hepes, pH 7, 1 mM dithiothreitol, 0.01% Brij35, 2 mM
MnCl2) plus 0.5 µCi of [-33P]ATP
(Amersham Biosciences). Various concentrations of staurosporine (Sigma)
up to 10 µM were added to test its inhibitory effect. The
reaction was conducted for 30 min at room temperature and stopped by
the addition of SDS-PAGE sample buffer plus EDTA (25 mM
final concentration). Ten microliters of the reaction were subjected to
electrophoresis. The reaction products were separated on an SDS-12%
polyacrylamide gel, and the radiolabeled myelin basic protein was
visualized after autoradiography of the dried gel.
Protein Crystallization-- Hanging droplets containing 1 µl of protein at 5 mg/ml and 1 µl of buffer (0.1 M Hepes, pH 7.5, 30 mM MgCl2, 150 µM AMP-PCP, 27% polyethylene glycol 400, 4% 1,3-butanediol) were equilibrated at 19 °C in Limbro plates against 1 ml of the same buffer but without the nucleotide and the butanediol. Diamond-like plates of 0.2-0.4 × 0.05-0.1 mm showed up after 2-3 weeks depending on preparation batches.
X-ray Diffraction and Crystal Structure Analysis--
X-ray
diffraction data sets (Table II) were collected from vitrified
crystals at 100 K using synchrotron radiation at the European
Synchrotron Radiation Facility (Grenoble, France) beamline ID14.2,
indexed and integrated using MOSFLM (15), and reduced with SCALA (16).
All crystals had C2221 symmetry with cell constants a = 114.5 Å, b = 122.0 Å,
c = 47.0 Å. The structure was solved by the molecular
replacement method using AMoRe (17) with coordinates of a polyserine
model derived from the crystal structure of rabbit phosphorylase kinase
(Protein Data Bank code 1PHK). An R-factor of 49.4% (for
the 12-4.5-Å resolution range) was calculated for the solution.
Simulated annealing using the program CNS (18) produced a significantly
better model (R-factor, 44.9%;
Rfree, 51.8%; 18-2.7 Å) from which manual
rebuilding, using XtalView (19), was possible. At this point the maps
showed unambiguous density for the nucleotide. Further refinement was
carried out using REFMAC (16). The final model displays an
R-factor of 19.1% and Rfree of
23.0% and includes residues 3-163 and 180-278, the AMP-PCP
nucleotide, two magnesium cations, and 85 water molecules (Table
II). There is no supporting electron density, and hence no coordinates
have been provided, for the His tag residues and for 16 residues
(164-179) comprised in the so-called activation and "P + 1" loops.
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RESULTS |
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Overall Structure--
The structure of the catalytic domain of
M. tuberculosis PknB was solved by molecular replacement and
refined at 2.2-Å resolution (Table II).
The final model accounts for 260 of 278 residues and displays good
main-chain stereochemical parameters for all but one amino acid, Ala-44
(see below). The overall fold of the catalytic domain of PknB is
similar (Fig. 1, B and C, and Table I) to that of
the eukaryotic protein kinases and consists of two lobes: an N-terminal
subdomain, including a curled -sheet and a long
-helix (
C),
and a C-terminal lobe, essentially composed by
-helices (for a
review, see Ref. 20). Protein kinases can be found in two
conformational states referred to as "open" and
"closed" conformations (21) depending on relative orientation
between both lobes. With some variations, the closed conformation
correlates with the active state of these enzymes. The movements that
bring one conformation into the other involve two main hinge points,
one in the Gly-rich loop and the other in the link region between the
two lobes (Fig. 1B). In the
structure that we report here, the catalytic domain of PknB adopts an
overall closed conformation with the nucleotide tightly bound in the
deep cleft between the N-terminal and the C-terminal lobes (Fig.
1B). This is in contrast with PKA (22), which was found in
the closed conformation only for the ternary (enzyme-nucleotide-substrate) complex but in agreement with what has
been observed for the binary enzyme-nucleotide complex of PhK (Ref.
23 and see Fig. 1C).
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One of the characteristics that define the closed conformation of a
protein kinase is the relative orientation of its C helix. In the
closed state, the position of this helix is such that it allows a
crucial contact to be established between a lysine residue in the
3
strand and a glutamic acid in the
C helix. As a result, the
lysine is appropriately oriented to position the
- and
-phosphates of the nucleotide for catalysis. Fig. 1C
shows that the N terminus of the
C helix of both PknB and PhK does
not match the position of the same region in PKA. However, PknB Lys-40
(PKA Lys-72) is hydrogen-bonded to Glu-59 (PKA Glu-91), and the lysine
does interact with the
- and
-phosphates of the nucleotide.
Therefore, as for PhK, the orientation of PknB
C helix can be
described as corresponding to a closed conformation of the enzyme.
In the crystal structure of the unphosphorylated form of the
D1161A mutant of IRK (24) the enzyme is in an overall closed conformation, but the spatial disposition of the C helix prevents the Lys-Glu contact. The authors (24) propose that the
phenylalanine in the Asp-Phe-Gly motif (residues 156-158 in PknB) of
the magnesium-positioning loop (Fig. 1, A and B)
is responsible for either allowing or blocking the movement that brings
the
C helix to its active position. In PknB, this phenylalanine
is in a conformation similar to that found in PKA and, as stated above,
allows the Lys-Glu contact even if the
C helix disposition is not
the same as that observed in PKA (Fig. 1C). Maintaining the
active conformation of the
C helix in PknB may be at the cost of
some strain and disorder at its N terminus, namely in the loop that
connects it to the
3 strand. This could explain the abnormal
(
,
) angles of the Ala-44 amino acid and the poor density observed
in the side chain of Arg-48 (Fig. 2).
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There is another difference in the N-terminal lobe between PknB and
both PhK and PKA. It results from the insertion in PknB of four amino
acids between the 4 and
5 strands (Fig. 1). This makes the PknB
N-terminal
-sheet more curled than in other kinases, a feature that
might be important for the interaction of PknB with other factors or
with its own juxtamembrane linker.
Finally it is worth mentioning that Arg-101, a residue in the hinge region between the lobes, occludes part of the groove that, in PKA, is filled by the peptide substrate. This is suggestive of a particular mode of substrate recognition and may be of great interest in the design of PknB inhibitors.
Nucleotide Binding--
The Gly-rich motif in the N-terminal lobe
of STPKs and PTKs acts as a flap that either prevents the binding of or
firmly covers the nucleotide (25). Like PhK, PknB differs from
the consensus sequence of this motif
(GX1GX2GX3V)
in that the third glycine is replaced with a serine (Fig.
1A). Moreover, a methionine residue, instead of the usual
tyrosine or phenylalanine, occupies the hydrophobic
position.
Nevertheless, variations in this motif are not uncommon and may play a
functional role. Interestingly PknB has a glycine at the
X2 position, which gives increased flexibility
to the external lip of the flap. This may facilitate a closer packing
of the nucleotide
-phosphate compared with the structure of other
kinases poised for phosphorylation such as PKA (Refs. 26 and 27, and
see Fig. 3). Thus, the main-chain amides
of Gly-21, Met-22, and Ser-23 bind only the
-phosphoryl group (Fig.
3B) and not the
- and
-phosphates as in the closed
conformation of PKA (26, 27).
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The adenine moiety is buried in a hydrophobic pocket (Fig.
3A) and makes two hydrogen bonds with the enzyme: its
N-6 position binds the main-chain oxygen of Glu-93, and the N-1
atom binds the amide of Val-91. The ribose is more loosely bound to the
enzyme than in PKA or PhK: its O-3* position binds the backbone
carboxyl of Ala-142, but the O-2* atom is not in direct contact with
the protein. The nucleotide is in the anti conformation
( =
154°), and the ribose bears a C3'-endo
puckering. As mentioned above, an important difference between PknB and
other kinases co-crystallized with nucleotide triphosphates involves
the position of the
-phosphate. Whereas the angle displayed by the
three phosphates in PKA (Protein Data Bank code 1ATP) is 105°, it is
162° in PknB, resulting in an almost linear triphosphoryl group.
Two metal ions, identified as magnesium and displaying well defined
octahedral coordination spheres, are found in the structure of PknB
(Fig. 3). In PKA, a "primary" activating cation binds the - and
-phosphates as well as Asp-184. In PknB, the equivalent magnesium is
shifted 2.5 Å toward the small lobe, consistent with the new location
of the
-phosphate, and binds the same phosphoryl groups but not
Asp-156 (PKA Asp-184). On the other hand, in PKA a "secondary"
cation binds more weakly the
- and
-phosphates as well as Asn-171
(26), and in some structures, it also binds Asp-184 (27). This second
metal ion has an inhibitory role, probably by slowing down the release
of the ADP product, which is the rate-limiting step in the kinase
reaction. One magnesium ion occupies the same location in PknB, but it
coordinates the
- and
-phosphates and both Asn-143 (PKA Asn-171)
and Asp-156.
Active Site Residues--
When the structure of PknB is superposed
onto the active (closed) conformation of PKA it becomes apparent that
all the amino acids in the active site are correctly positioned to
phosphorylate a putative substrate (Fig.
4). Thus, the backbone oxygen of Asp-138 makes a hydrogen bond with the amide nitrogen of Asn-143. This interaction places Asp-138 in a suitable position to orientate an
attacking hydroxyl from a putative substrate. Another important residue
for maintaining the active conformation of the catalytic loop (residues
135-143) is Asp-198. This amino acid is hydrogen-bonded to the
backbone amide groups of His-136 and Arg-137 and to the N2 of His-130, thus becoming a central pivot in this
delicate network of interactions (Fig. 4).
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However, the nucleotide -phosphate (Fig. 4) is far from Asp-138 (PKA
Asp-166), the proposed (although controversial) catalytic base (28),
and from Lys-140 (PKA Lys-68), a residue highly conserved among STPKs
but not in PTKs. Possibly due to the lack of interaction with the
-phosphate, the N
atom of Lys-140 is disordered in
this PknB structure.
The Activation and P + 1 Loops--
The activation loop of protein
kinases (residues 164-177 in PknB) encompasses the region between the
9 strand, just next to the magnesium-positioning loop, and the P + 1 loop (see below). The activation loop can undergo large conformational
changes (29) that may determine the catalytic state of the enzyme.
Fourteen residues in the activation loop of PknB, from Ala-164 to
Ile-177, are not visible in this structure and hence are presumably disordered.
The so-called P + 1 loop (PknB residues 178-183) follows the C
terminus of the activation loop and helps to accommodate the P + 1 substrate residue. It is thought to play a major role in the
discrimination between serine/threonine and tyrosine amino acids since
it governs the distance between the substrate backbone and the active
site (30, 31). As expected for an STPK (31), this loop contains a
threonine, Thr-179, rather than the proline found at this position in
PTKs. The first two residues of the P + 1 loop (Gly-178 and Thr-179)
are disordered in the PknB structure.
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DISCUSSION |
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PknB Is Poised for Phosphotransfer but Binds the Nucleotide in an
Unusual Conformation--
The deviation of the -phosphate position
toward the N-terminal lobe observed in PknB is new, albeit diversity in
the position of this phosphate has been described for other kinases.
For instance, in the ternary complex of IRK (Protein Data Bank code
1IR3) this phosphate, and to a lesser extent the
-phosphate,
protrudes out of the cleft, lying 5 Å away from the putative
attacking hydroxyl.
The most interesting trait in the interaction between PknB and the
nucleotide is that the active site amino acids of the enzyme appear to
be ready for phosphotransfer, but the -phosphate of the nucleotide
is far from its functional place (Fig. 4). This differs from other
binary nucleotide-kinase complexes (31), namely
cyclin-dependent kinase 2, hematopoietic cell kinase,
mitogen-activated protein kinase, and fibroblast growth factor receptor
kinase, which were captured in conformations different from the fully active state and exhibit different protein residues out of their functional position. Although it is possible that the use of AMP-PCP instead of ATP in the crystallization assays may have produced this
peculiar conformation of the triphosphoryl group in PknB, it is worth
noting that at least another kinase, the insulin receptor kinase
(Protein Data Bank code 1I44), has been crystallized in complex with
the same analog, and yet the nucleotide displays a standard conformation.
PknB Is Not Inhibited by Staurosporine--
The analysis
of the nucleotide-binding pocket may prove useful in the
characterization and design of potentially inhibitory drugs.
Staurosporine is a wide range inhibitor of protein kinases with
nanomolar affinity for many of them (e.g. PKA) but only
micromolar potency against kinases such as casein kinase 1, casein
kinase 2, mitogen-activated protein kinase, and the C-terminal Src
kinase (32). It has been suggested (33) that the loss of a hydrogen bond between the N-31 atom of staurosporine and the residue
structurally equivalent to PKA Asp-127 may partially explain this
difference. In PknB this amino acid, Thr-99, binds the amide backbone
of Asp-102. The same situation has been reported in casein kinase 1, casein kinase 2, and C-terminal Src kinase (33). As a result, the
hydroxyl group of Thr-99 is in an orientation that would prevent
its interaction with staurosporine N-31. This hypothesis is in
agreement with the almost null inhibitory effect of this drug upon PknB
(Fig. 5).
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The Activation Loop of PknB Is Structurally Disordered--
The
activation loop plays a critical role in the control of the activity of
protein kinases as well as in the recognition of their substrates. In
some cases (e.g. IRK) this loop can adopt a conformation,
referred to as "gate-closed" (24), that impedes the binding of both
the nucleotide coenzyme and the peptide substrate. There are also
examples, such as mitogen-activated protein kinase, in which a
particular conformation of this loop is required to establish the
appropriate orientation of the C helix that allows the binding of
ATP. In cyclin-dependent kinase 2 these two control mechanisms co-exist. A general way to regulate the conformation of this
loop consists in the phosphorylation of one or more of its amino acids.
With few exceptions, phosphorylation of the activation loop constitutes
one of the key regulatory mechanisms that increase the catalytic
activity of PTKs (34). In the structure of PknB, most of the activation
loop is disordered, including various serine and threonine residues
that could be suitable targets for phosphorylation. Indeed disorder in
the activation loop is not rare in inactive or partially active kinase
structures. However, we have demonstrated that this PknB construction
is active (Fig. 5). In this respect, it has been suggested (35) that it
might be a general feature of the steric autoinhibition of protein
kinases that the activation loops become disordered when their
gate-closed conformation is lost. An example can be found in the
structure of the D1161A mutant of IRK in complex with AMP-PCP (24). In
this case, the mutation displaces the equilibrium of the activation
loop in the unphosphorylated enzyme toward a disordered "open gate"
conformation. This facilitates the nucleotide binding without affecting
the binding of the peptide substrate. In some cases the complete
phosphorylation of the activation loop is sufficient to fix its open
gate conformation, whereas in other kinases the presence of the peptide
substrate is also required to attain its full stabilization through an
induced-fit mechanism (33). Thus, the actual phosphorylation state of
the activation loop or the absence of its peptide substrate could explain the observed disorder in PknB. Nevertheless, other explanations could also be considered, such as a stabilizing role of the
juxtamembrane linker (absent in this PknB construction) or some
putative modulator factor(s).
In PKA, a phosphate-binding pocket accommodates the phosphorylated
Thr-197 from the activation loop. A similar role could be played in
PknB by three arginines: Arg-55, Arg-137, and Arg-161. The side chains
of these three residues are disordered, especially in Arg-55 and
Arg-161. Another arginine, Arg-58, is also close to this cluster,
raising the possibility of a larger positively charged pocket in PknB.
It has been proposed (20) that in PKA the interaction between Arg-165
(PknB Arg-137) next to the catalytic loop and the phosphate bound to
Thr-197 would place the Asp-Phe-Gly motif in the activation loop into
proper orientation for catalysis. In PknB, the disorder of the Arg-137
side chain does not preclude the correct position of the Asp-Phe-Gly
motif. According to one hypothesis (23), the proper location of this
motif would permit the C helix to occupy its active conformation.
Thus, in PknB the active position of the
C helix appears to be
dissociated from a stable interaction between Arg-137 in the catalytic
loop and a phosphoresidue in the activation loop.
Substrate Binding--
Protein substrates of PknB have yet to be
identified. However, some clues about substrate specificity can be
drawn from the comparison with the structures of other STPKs
crystallized with their peptide substrates. For instance, Glu-208
occupies approximately the same position as PKA Glu-230, a residue
involved in the recognition of the P 2 amino acid (an
arginine in the PKA recognition sequence consensus,
RRXSI, where the Ser residue is the
phosphorylation site). Another residue, Tyr-182 (PKA Tyr-204)
could also stabilize a positively charged amino acid at the P
2 position. However, only Glu-208 is conserved among the four glutamates
involved in the PKA recognition of the two arginines at positions P
2 and P
3, suggesting that PknB might have a lower preference for basic amino acids at these positions. On the other hand, the chemical composition of the disordered part of the P + 1 loop as well as the
hydrophobic surface defined by residues Ile-159, Ala-180, Leu-183, and
Ala-188 suggest that a hydrophobic residue could constitute a good
candidate for the P + 1 amino acid.
Conclusions--
This work confirms the remarkable fold
conservation of the Ser/Thr/Tyr protein kinase superfamily among
organisms belonging to phyla with evolutionary histories as distant as
eubacteria and high eukaryotes. Nevertheless, the structure of the
catalytic core of PknB presents some peculiarities. Namely the binding
of the nucleotide and the interplay of the different regions involved in the activity and regulation of the enzyme show some variations with
respect to the eukaryotic homologues of known structure, enriching our
comprehension about the fine tuning of the proteins belonging to this
superfamily. Further work, aimed at the identification of the
substrates of PknB, aside from its utmost biological interest, may help
to define the role played by the activation loop of the enzyme and
hence disclose the molecular mechanism of regulation of this
mycobacterial protein kinase.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. T. Cole for kindly providing us with the MTCY10H4 cosmid. We also acknowledge the European Synchrotron Radiation Facility at Grenoble for technical assistance and access to their installations.
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FOOTNOTES |
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* This work was funded by the Génopole Program; the Institut Pasteur (Programme Transversal de Recherche No. 46); the Programme de Recherches Fondamentales en Microbiologie, Maladies Infectieuses et Parasitologie from the Ministère de l'Education Nationale de la Recherche et de la Technologie; and the European Community (Grant QLRT-2000-02018).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1O6Y) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Recipient of a Federation of European Biochemical
Societies long term fellowship. To whom correspondence may be
addressed: York Structural Biology Laboratory, Dept. of Chemistry,
University of York, York YO10 5YW, UK. Tel.: 44-1904-328276; Fax:
44-1904-328266; E-mail: mol@ysbl.york.ac.uk.
§ Present address: Laboratoire de Chimie Bactérienne, CNRS, 31 chemin J. Aiguier, 13402 Marseille cedex 20, France.
¶ To whom correspondence may be addressed: Unité de Biochimie Structurale, Institut Pasteur, 25 rue du Dr. Roux, 75724, Paris cedex 15, France. Tel.: 33-1-4568-8607; Fax: 33-1-4568-8604; E-mail: alzari@pasteur.fr.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M300660200
2 B. Boitel, M. Ortiz-Lombardía, F. Pompeo, and P. M. Alzari, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
STPK, serine/threonine protein kinase;
AMP-PCP, adenosine
5'-(,
-methylene)triphosphate;
IRK, insulin receptor kinase;
PhK, phosphorylase kinase;
PKA, cAMP-dependent protein kinase;
PTK, protein-tyrosine kinase.
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