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. Author manuscript; available in PMC: 2010 Dec 1.
Published in final edited form as: Curr Opin Struct Biol. 2009 Nov 14;19(6):650–657. doi: 10.1016/j.sbi.2009.10.017

Signaling mechanisms of the Mycobacterium tuberculosis receptor Ser/Thr protein kinases

Tom Alber 1
PMCID: PMC2790423  NIHMSID: NIHMS156582  PMID: 19914822

Abstract

Like eukaryotes, bacteria express receptor Ser/Thr protein kinases (STPKs) that initiate a wide variety of signaling networks. Recent biochemical and structural studies of the STPKs of Mycobacterium tuberculosis have revealed that bacterial and eukaryotic STPKs adopt common folds and share mechanisms of substrate recognition and regulation. Mycobacterial receptor STPKs are activated by dimerization though two distinct interfaces that promote activation-loop phosphorylation. The active STPKs phosphorylate diverse substrates within the bacterial cell, including other kinases as well as proteins involved in many central physiological processes. In the case of the FHA-domain protein, GarA, the unphosphorylated protein regulates primary metabolism, while phosphorylation mediates GarA autoinhibition. These studies have begun to define the activation mechanisms and the biological regulatory functions of the mycobacterial STPKs.

Keywords: eukaryotic-like kinase, activation mechanism, substrate specificity, autoinhibition, allosteric control, PknB

In all three kingdoms of life, receptor protein kinases play essential roles regulating cell physiology in response to extracellular cues. While two-component systems shuttle chemically labile phosphoryl groups between His and Asp residues, stable phosphorylation that is reversed by cognate phosphatases is mediated by the spatial and temporal regulation of Ser/Thr/Tyr kinases. With more than 10,000 members [1], this protein superfamily is among the largest known. Long associated exclusively with eukaryotes, STPKs were discovered in the last 17 years in bacteria, including many pathogens.

These “eukaryotic-like” STPKs play essential roles in growth, virulence, persistence and reactivation, generally by signaling within bacterial cells. In Pseudomonas aeruginosa, for example, Thr phosphorylation by the PpkA kinase regulates assembly of the Type VI secretion system that influences virulence in clinical isolates [2, 3]. The Bacillus kinase PrkC mediates reactivation of spores in response to fragments of the peptidoglycan [4]. Mycobacterium tuberculosis (Mtb) expresses 11 STPKs (PknA-PknL), nine of which are predicted transmembrane receptors with the kinase domain located within the mycobacterial cell [5]. Although the soluble Mtb kinase, PknG, has been reported to be secreted by Mtb and play an essential role in signaling in host cells [6], this model has not been corroborated by subsequent studies. To the contrary, biochemical studies, the occurrence of orthologs in nonpathogenic bacteria, and characterization of pknG knockout mutants of Mtb and Corynebacterium glutamicum show that PknG instead controls primary nitrogen and carbon metabolism within bacterial cells [7, 8, 9].

In Mtb, PknA and PknB are essential for growth [10, 11, 12]. Although Mtb PknB and Bacillus PrkC are orthologous kinases, the deletion of prkC results in viable bacteria that are defective in reactivation of spores [4]. These distinct phenotypes indicate that STPKs containing similar extracellular ligand-binding domains can be “wired” to cell physiology in distinct ways in different bacterial species. Here I survey recent work aimed at understanding the fundamental mechanisms of regulation and substrate recognition of the Mtb STPKs. Because these kinases show a maximum of 30% sequence identity to human homologs, studies of the bacterial STPKs inform not only the general mechanisms of signaling, they also provide important foundations for developing new antibiotics.

Conserved STPK structures

The Ser/Thr/Tyr kinases are molecular switches. Many structural features in the shared STPK fold influence the equilibrium between more active (“on”) and less active (“off”) states [13, 14]. Nearly universally, the Ser/Thr/Tyr protein kinases exist constitutively in the off state, and signals relieve autoinhibition. In contrast to the activated conformations, in which the catalytic machinery is assembled similarly, the inhibited states are structurally diverse. Several structural elements converge at the active site to control ATP binding, protein-substrate binding and the alignment of the catalytic groups.

A common chemical mechanism for activation involves phosphorylation of one or more sites in the activation loop, a centrally located, 15-33-residue segment flanked by the DFG and APE sequence motifs [13, 14]. In the off state, the activation loop generally blocks the active site or remains flexible. In contrast, the phosphorylated activation loops adopt a distinct conformation that assembles a pThr binding site that stabilizes the active conformation. This conformational rearrangement permits ATP access, brings together dispersed elements of the catalytic machinery and forms part of the protein-substrate binding site.

The first crystal structures of a bacterial STPK kinase domain (KD), showing the phosphorylated Mtb PknB KD bound to two different ATP analogs [15, 16], displayed features largely characteristic of active eukaryotic homologs (Fig. 1). The nucleotide was sandwiched between the N- and C-lobes, engaging the glycine-rich P-loop, a signature β-hairpin with main-chain amides that coordinate the triphosphate moiety. The Lys40-Glu59 ion pair bridged the nucleotide and the C-helix. While the position of the C-helix was reminiscent of inactive c-Src, active eukaryotic kinase structures with the C-helix position resembling PknB also have been reported [13, 14]. Thus, the crystal structure of PknB is thought to largely define the active state of bacterial STPKs.

Figure 1. The PknB KD:ATPγS structure resembles the transition-state analog complex of cAMP-dependent protein kinase (PKA).

Figure 1

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Superimposed backbone ribbons show the similarity of the classic kinase folds of PknB (blue) and PKA (yellow), including the two-domain structure and the P-loop covering the nucleotide. The bound nucleotides and several active-site residues in PknB:ATP-γ-S (blue) and the PKA:ADP:AlF3:peptide transition-state-analog complex [45] shows that conserved residues contact the nucleotides in similar conformations.

Unlike a typical active STPK, however, the PknB activation loop was disordered. This disorder remains a puzzle, because the PknB KD was phosphorylated on 3–7 sites [15], including both Thr residues in the activation loop essential for activation [17]. With the exception of PknG (which is not switched on by activation-loop phosphorylation), this functional phosphorylation pattern is conserved in mycobacterial receptor kinases [18, 19, 20, 21]. Inhibiting PknD autophosphorylation with a small molecule in Mtb limited phosphorylation of cellular proteins, showing that STPK autophosphorylation is a central regulatory strategy in vivo [22]. Nonetheless, a disordered activation loop also was found in the structure of phosphorylated apo-PknE KD [23], suggesting that phosphorylation may not stabilize a unique active structure. However, heterogeneous or hyper-phosphorylation may stabilize multiple conformations. Alternatively, binding to an unidentified cofactor or substrates may be required to fold the activation loop. Ultimately, understanding the changes caused by activation-loop phosphorylation will require structural characterization of an unphosphorylated KD to compare the off state directly to the phosphorylated form.

Activation mechanism

Current evidence indicates that the Mtb receptor kinases are activated by reversible interactions through two distinct protein interfaces. These contacts function to promote phosphorylation of the activation loop by different mechanisms. Although the phosphorylated KDs of PknB and PknE removed from the membrane are monomeric at typical concentrations in solution (Kd values in the low mM range), they crystallized as structurally similar dimers (Fig.2) [15, 16, 23]. Disparate observations indicate that these “back-to-back” dimers stabilize the active conformation. The N-lobe dimer interface is conserved in orthologs of PknB. Rapamycin-promoted dimerization of unphosphorylated PknD and PknB KD fusion proteins to the FK506 binding protein and FKBP12-rapamycin-associated protein (FRAP) stimulated phosphorylation activity [24]. Mutations in the N-lobe dimer interface reduced this activation, limited autophosphorylation and altered substrate specificity. In contrast, dimerization with an inactive, catalytic-site mutant stimulated the wild-type PknD KD in vitro and in vivo. Because the inactive subunit in this heterodimer is incapable of phosphorylating the paired wild-type subunit, these results supported the idea that dimer formation allosterically activates unphosphorylated PknD [24]. The generality of this activation was demonstrated recently in Pseudomonas aeruginosa, where insertion of FK506 binding protein into an extracellular loop resulted in a PpkA kinase that was activated in vivo by addition of a small-molecule dimerizer [3]. Thus, N-lobe dimerization activates different bacterial receptor kinases in vitro and/or in vivo.

Figure 2. Mtb PknB, PknE and human PKR KDs form structurally similar activated dimmers.

Figure 2

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Ribbon drawings of PknB-ATPγS (left; C-helix yellow, nucleotide in spheres), apo-PknE (middle), and a superposition (right) of PknB (blue) and PKR (orange) reveal the conserved architecture. The N-lobe interaction holds the catalytic sites away from each other and activates the unphosphorylated kinases by an allosteric mechanism. The back-to-back interface communicates to the active site over 25 Å across the protein.

Remarkably, the back-to-back N-lobe interface also mediates allosteric activation of human PKR, the cytosolic double-stranded RNA-dependent protein kinase [25], and Ire1, the transmembrane kinase that mediates the unfolded protein response [26]. While these KD dimmers stabilize the active conformations, the Ire1 dimer also mediates assembly of a larger oligomer that stimulates the cleavage of physiological substrates by the appended ribonuclease domains [27]. The conservation of a structurally analogous active dimer in two Mtb STPKs and in human PKR implies that this mechanism of protein kinase regulation is ancient and broadly distributed.

It is noteworthy that the kinases thus far found to form back-to-back, N-lobe dimers respond to ancient molecules, including double-stranded RNA (PKR), unfolded proteins (Ire1), and potentially for the bacterial kinases, peptidoglycan fragments (PknB) and disulfide bonds (PknE). In the phylogenetic tree of the human kinome, PKR and Ire1 are not members of the major subfamilies, but instead diverged close to their common ancestor [28]. Combined with the distribution of PknB in diverse bacteria, the occurrence of STPKs in all three kingdoms of life, and the apparently ancient divergence of mycobacterial and human STPKs, these sequence relationships support the idea that the bacterial STPKs arose by divergent evolution.

The idea that the N-lobe interface stabilizes the active conformation left open the question of how the kinase domains are autophosphorylated. This is a general question in protein kinase biochemistry, and both true intramolecular autophosphorylation and intermolecular reactions have been detected. For intermolecular (trans) autophosphorylation, it is unknown if the sequence around the phosphorylation sites or structural interfaces between the kinase molecules mediate recognition.

Structural studies of PknB implicated a second interface--including the G-helix and activation loop in the C-lobe--in bacterial KD autophosphorylation. The structure of an ATP-competitive inhibitor bound a PknB KD variant [29] unexpectedly revealed features consistent with an activation complex (Fig. 3). The inhibitor-bound PknB KD formed an asymmetric dimer, with the G-helix and the ordered activation loop of one KD in contact with the G-helix of the other. The activation loop of this putative “substrate” KD was disordered, with the ends positioned at the entrance to the partner KD active site. As expected for an ES complex, single amino-acid substitutions in the G-helix interface reduced activation-loop phosphorylation, and multiple replacements abolished KD phosphorylation and kinase activation. The unaltered consensus PknB phosphorylation motif (TQXϕϕ) [11] in the activation loop of the mutants was insufficient to mediate autophosphorylation. These results support a model in which a structurally asymmetric, “front-to-front” association through the G-helix mediates intermolecular autophosphorylation of PknB and homologous kinases.

Fig. 3. Inhibitor complex of a PknB KD mutant resembled an autophosphorylation complex.

Fig. 3

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A. PknB monomers formed an asymmetric offset dimer with contacts between the G-helices. Identical interface residues occur in distinct environments in the presumptive substrate (yellow) and enzymatic (blue). The activation loop was disordered in the substrate KD (yellow dashed line) and ordered (red line) in the enzyme KD.) B. Surface representation showing the contacts between kinase molecules and the ordered activation loop (red) in the enzyme KD.

Once phosphorylated, the monomeric Mtb PknD KD is fully active in vitro, and dimerization does not further stimulate kinase activity [24]. This property provides a mechanism to propagate and amplify an activation signal to STPK molecules than are not engaged by the extracellular ligand [29] (Fig. 4). In addition, phosphorylation in trans allows a signal sensed by one STPK to activate another. Mtb PknB phosphorylates PknA, for example, and may activate PknA to phosphorylate distinct substrates [11]. Such crosstalk between STPKs provides a mechanism to integrate pathways, but also raises the problem of establishing signaling specificity. The generality of crosstalk, the specific networks of the Mtb STPKs, and the impacts of cross phosphorylation have yet to be defined.

Fig. 4. Model for Mtb receptor STPK regulation [24].

Fig. 4

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The inactive, unphosphorylated kinase monomer (left) is paired by an extracellular signal. The dimer adopts a conformation that is activated for autophosphorylation and substrate phosphorylation. The autophosphorylated monomers are active and amplify the signal (right). After the signal dissipates, the PstP phosphatase returns the kinase to the unphosphorylated inactive form.

Substrate specificity and metabolic control

The biological roles of the STPKs depend crucially on their substrate specificity. Recent studies demonstrate that the Mtb STPKs phosphorylate proteins involved in a large variety of processes, including cell metabolism, transcriptional regulation, translation, cell-wall biosynthesis, and cell division. Demonstrating the functional significance of observed phosphorylations remains an ongoing challenge. A review tabulating reported STPK substrates in Mtb was published recently [9], and I will not reprise that list here. A varied combination of in vitro and in vivo experiments has proven essential to make progress. Biochemical methods, for example, have been critical to identify candidate substrates, quantify modifications, map phosphorylation sites and demonstrate large changes in activity upon phosphorylation. At the same time, studies of the effects of phosphorylation in vivo have been critical to establish the biological functions of STPK regulation.

Pioneering work by Husson and coworkers identified Wag31, Rv1422 and PknA as heterologous substrates of mycobacterial PknB [11]. Wag31 is dramatically localized to the cell poles [30], raising the question of whether the STPK shows a similarly restricted spatial distribution. PknD and PknB respectively phosphorylate the σ-factor regulators Rv0516c and RshA [24] on noncanonical sites that do not fit the current paradigm for σ-factor regulation. Nonetheless, RshA phosphorylation was shown recently to regulate SigH in vivo [31]. PknD activation in Mtb also altered the expression of more than 100 genes regulated by SigF, providing evidence that PknD converts environmental signals into a transcriptional response [22]. Purified Mtb STPK KDs except those of PknG, PknI and PknJ phosphorylated the FAS-II components KasA and KasB in vitro, and these fatty-acid biosynthetic enzymes are phosphorylated in M. bovis BCG [32]. Nearly all the Mtb STPKs phosphorylated GarA in vitro (see below) [33]. Some reported substrates, however, present unresolved paradoxes. The extracellular cell-wall transpeptidase, PbpA, for example, has been reported to be a substrate of PknB [34], even though the kinase, ATP and the antagonistic phosphatase, PstP, are intracellular. Similarly, the reported PknD phosphorylation site on MmpL7 [35] is predicted to be extracellular. Overall, many substrates remain to be discovered, and the mechanisms of protein-substrate recognition remain to be determined.

Proteins containing forkhead-associated (FHA) domains comprise an important class of Mtb STPK substrates. FHA domains bind pThr peptides [36], triggering assembly of protein complexes. Mtb produces five FHA-domain proteins: EmbR, GarA, an ABC transporter (Rv1747), and two proteins (Rv0019c and Rv0020c) co-expressed with PknA and PknB. Molle and coworkers identified the transcriptional regulator, EmbR, as the first cognate substrate of a Mtb STPK [20]. This phosphorylation stimulates promoter DNA binding in vitro and activates transcription of the embCAB genes (involved in arabinan biosynthesis) in vivo [37]. PknH autophosphorylation was essential to bind and phosphorylate EmbR, but PknH phosphopeptides, including a phosphorylated activation-loop sequence, failed to bind to the EmbR FHA domain [38]. Indeed, the cocrystal structure of EmbR showed a noncognate pThr peptide from yeast Rad53 bound to the Mtb FHA domain. This structure confirmed the canonical mechanism of pThr recognition [38], but left open the nature of the determinants of binding specificity.

In general, a complex network of STPKs phosphorylates the Mtb FHA-domain proteins in vitro [39]. Alzari and coworkers established the FHA-domain protein GarA as an efficient substrate of several Mtb KDs, with phosphorylation occurring at Thr21 (PknG) or Thr22 (other STPKs) in a disordered N-terminal extension [7, 33]’. Binding of the GarA FHA domain to the PknB KD required the phosphorylated kinase activation loop [33]. Because this interaction would be expected to sequester the activation loop away from the active conformation, however, the basis for phosphorylation of the FHA-domain protein in such a complex remains enigmatic. In contrast, autophosphorylation of sites in the first 65 amino acids of PknG, N-terminal to the kinase domain, recruits GarA and enhances phosphorylation [7].

Exciting recent papers from several groups established not only a new mechanism of GarA regulation of carbon and nitrogen metabolism, but also showed that Thr phosphorylation autoinhibits GarA (Fig. 5). Affinity chromatography of mycobacterial extracts identified NAD+− dependent glutamate dehydrogenase (GDH), α-ketoglutarate decarboxylase (KGD), the α subunit of glutamine synthetase (GS), and PknG as soluble GarA binding partners [7, 8]. Although it contains a pThr-binding FHA domain, GarA unexpectedly recognized the unphosphorylated forms of GDH, KGD and GS. The pThr-binding site functions instead to recognize phosphorylated STPKs and, once the N-terminal tail is phosphorylated on Thr21 or Thr22, to stabilize the tail in a conformation that inhibits target-enzyme binding [8, 40].

Figure 5. Phosphorylation autoinhibits GarA.

Figure 5

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A. Schematic model of Mtb GarA inhibition [8]. The FHA domain (orange) engages the N-terminal tail (red) phosphorylated on Thr21 (by PknG) or Thr22 (by PknB), stabilizing a conformation that blocks binding of the STPKs and target enzymes GDH, KGD and GS. The N-terminal tail adopts many conformations in the on state. B. Ribbon diagram of the average NMR structure of GarA [8] showing the N-terminal tail (red) occluding the enzyme-binding surface. Spheres show pThr22 (atom colors) and sites where mutations block binding of all three (blue) or two of the three (light blue) unphosphorylated target enzymes. C. Superposition of the average NMR structures of OdhI [41], the GarA homolog in C. glutamicum, unphosphorylated (cyan) and phosphorylated on Thr15 (blue). Folding of the N-terminal tail into the pThr binding site causes little change in the core FHA domain structure.

Incisive NMR studies of the unphosphorylated and PknB-phosphorylated forms of GarA and the C. glutamicum ortholog, OdhI, revealed that this intramolecular binding orders the phosphorylated tail and occludes overlapping surfaces required for binding to all the target enzymes [8, 41]. In vitro assays demonstrated that GarA binding inhibits the catalytic activities of purified GDH and KGD, and, at the same time, activates GS. These three enzymes act on α-ketoglutarate at the nexus of the TCA cycle and ammonia fixation, and the combined effects of GarA favor glutamate biosynthesis. These results provide a beautifully simple explanation for the increased levels of glutamate in pknG knockout bacteria (in which GarA is hyperactive) [7,8].

These studies begin to provide a mechanistic understanding of the biological effects of Ser/Thr phosphorylation in Mtb. STPK phosphorylation also exhibits varied downstream effects on many proteins lacking FHA domains. Phosphorylation of the transcription factor VirS (by PknK) [42] increases affinity for promoter DNA sequences, while phosphorylation of Rv2175c (by PknL) inhibits promoter DNA binding [43]. PknD phosphorylation of the anti-anti-sigma factor homolog, Rv0516c, decreases binding of Rv2638 (ref [22]). Phosphorylation (by PknA or PknF) of FabH, a β-ketoacyl carrier protein involved in mycolic acid biosynthesis, blocks the entrance to the substrate channel and inhibits the enzyme [44]. Structural studies of the phosphorylated substrates of the Mtb STPKs promise to continue to reveal diverse mechanisms of allosteric control.

Prospects

Rapid recent progress has begun to define the mechanisms of activation, the cellular substrates and the biologic functions of mycobacterial STPKs. Dimerization and phosphorylation regulate the Mtb STPKs in ways that recapitulate and inform the activation mechanisms of eukaryotic family members. While Mtb kinase substrates are being discovered rapidly, little is known about how STPKs recognize target proteins. Further studies are needed to test the roles of the phosphorylation-site sequence, structural interactions with cognate kinases and STPK localization in substrate recognition. Much effort is focused on defining how the STPKs regulate bacterial physiology. Recent work on the PknB ortholog (PrkC) in Bacillus subtilis has provided an exciting emerging hypothesis that peptidoglycan (PG) fragments activate PknB and regulate PG synthesis, cell growth and division [4]. With 11 STPKs, Mtb affords a tractable, medically important system to explore these fundamental issues.

Acknowledgments

I am indebted to my colleagues and coworkers including C. Baer, A. Cavazos, J. Cox, B. Delagoutte, N. Echols, J. Endrizzi, A. Falick, Y. Feng, E. M. Flynn, J. Fraser, L. Gay, C. Gee, M. Good, A. Greenstein, C. Grundner, J. Holton, A. Iavarone, D. King, T. N. Lombana, J. MacGurn, C. Mieczkowski, A. Moskaleva, H.-L. Ng, L. Prach, D. Prigozhin, K. Pullen, S. Smith, P. Sung, D. Tomkiel, and T. Young for making possible our studies of the Mtb STPKs. I thank Yossef Av-Gay, John Kuriyan and Susan Taylor for irreplaceable discussions of kinase structure and signaling mechanisms. The TB Structural Genomics Consortium provided essential encouragement and collaboration. NIH R01 GM70962 supported this work.

Footnotes

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