Crystal Structure of the Catalytic Domain of the PknB Serine/Threonine Kinase from Mycobacterium tuberculosis*

Miguel Ortiz-LombardíaDagger, Frédérique Pompeo§, Brigitte Boitel, and Pedro M. Alzari

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 gamma -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


                              
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Table I
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).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -helix (alpha 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-beta -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 [gamma -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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -sheet and a long alpha -helix (alpha C), and a C-terminal lobe, essentially composed by alpha -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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Table II
Crystallographic statistics


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Fig. 1.   Overall structure of the catalytic domain of PknB. A, structure-based multiple sequence alignment of the kinase domains of PknB, PKA, and PhK. The secondary structure elements of PknB have been delineated on top of the sequences; their names are based in the standard notation for PKA. The 11 conserved residues of the Ser/Thr/Tyr protein kinase superfamily are marked with an asterisk. A dashed line indicates the disordered part of the activation loop in PknB. The figure was prepared with ESPript (36). B, tertiary structure of PknB. The essential residues have been depicted onto the secondary structure of PknB, colored in violet for the N terminus, dark green for the hinge region, and red for the C terminus. The catalytic loop is shown in cyan, and the magnesium-positioning loop is shown in light green. The unseen part of the activation loop is represented as a dashed black line. C, superposition of the PknB structure (red) onto the closed conformation of PKA in the ternary complex (Protein Data Bank code 1ATP, green) and PhK in the binary nucleotide-enzyme complex (Protein Data Bank code 1PHK, yellow). The full kinase domain was used to drive the superposition. Figs. 1, B and C, 3B, and 4 were prepared with Molscript (37) and Raster3D (38).

One of the characteristics that define the closed conformation of a protein kinase is the relative orientation of its alpha 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 beta 3 strand and a glutamic acid in the alpha C helix. As a result, the lysine is appropriately oriented to position the alpha - and beta -phosphates of the nucleotide for catalysis. Fig. 1C shows that the N terminus of the alpha 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 alpha - and beta -phosphates of the nucleotide. Therefore, as for PhK, the orientation of PknB alpha 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 alpha 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 alpha 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 alpha C helix disposition is not the same as that observed in PKA (Fig. 1C). Maintaining the active conformation of the alpha 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 beta 3 strand. This could explain the abnormal (phi ,psi ) 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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Fig. 2.   Stereoview of the region surrounding Ala-44 in the loop connecting the beta 3 strand to the alpha C helix. This loop exhibits a 310 helical conformation. The sigmaA-weighted (2mFobs - DFcalc) electron density map has been contoured at 1.0 sigma  level around residues 41-52. This figure and Fig. 3A were made with XtalView (19) and Raster3D (38).

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 beta 4 and beta 5 strands (Fig. 1). This makes the PknB N-terminal beta -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 (GX1GX2phi GX3V) 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 phi  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 gamma -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 gamma -phosphoryl group (Fig. 3B) and not the beta - and gamma -phosphates as in the closed conformation of PKA (26, 27).


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Fig. 3.   Nucleotide binding. A, close stereoview of the nucleotide binding site showing the hydrophobic pocket occupied by the adenine moiety, the extended conformation of the triphosphoryl group, and the coordination of the two magnesium ions. The sigmaA-weighted (2mFobs - DFcalc) electron density map has been contoured at 1.1 sigma  level around the nucleotide. B, comparison of the nucleotide packing against the Gly-rich loop in PknB (left) and PKA (right). Both structures are shown in the same orientation after superposition as explained in the legend of Fig. 1C. Dotted lines indicate the hydrogen bonds made by the nucleotide with the Gly-rich loop and the divalent cations.

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 (chi  = -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 gamma -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 beta - and gamma -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 gamma -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 alpha - and gamma -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 alpha - and beta -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 Nepsilon 2 of His-130, thus becoming a central pivot in this delicate network of interactions (Fig. 4).


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Fig. 4.   Detail of the active site of PknB superimposed onto that of PKA (Protein Data Bank code 1ATP). For PknB the color code is as in Fig. 1B, while PKA is shown in green except for its nucleotide-bound divalent cations, which are in gray. Some PknB key residues in this zone are labeled, and their hydrogen bonds are indicated by dotted lines (see text for details). The position of the gamma -phosphate (gamma P) in PKA is also indicated (green label).

However, the nucleotide gamma -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 gamma -phosphate, the Nzeta 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 beta 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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PknB Is Poised for Phosphotransfer but Binds the Nucleotide in an Unusual Conformation-- The deviation of the gamma -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 beta -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 gamma -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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Fig. 5.   Effect of staurosporine on PknB kinase activity in a myelin basic protein (MBP) phosphorylation assay. PknB or PKA were incubated with the model substrate myelin basic protein in presence of [gamma -33P]ATP and various concentrations of staurosporine as indicated. The reaction products were resolved on an SDS-polyacrylamide gel that was dried and autoradiographed.

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 alpha 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 alpha C helix to occupy its active conformation. Thus, in PknB the active position of the alpha 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 - 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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/).

Dagger 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.

    ABBREVIATIONS

The abbreviations used are: STPK, serine/threonine protein kinase; AMP-PCP, adenosine 5'-(beta ,gamma -methylene)triphosphate; IRK, insulin receptor kinase; PhK, phosphorylase kinase; PKA, cAMP-dependent protein kinase; PTK, protein-tyrosine kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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