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Kinase Regulation in Mycobacterium tuberculosis: Variations on a Theme
Structure
Previews Kinase Regulation in Mycobacterium tuberculosis: Variations on a Theme Sina Reckel1 and Oliver Hantschel1,* 1Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, E ´ cole Polytechnique Fe´de´rale de Lausanne (EPFL), Lausanne 1015, Switzerland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2015.05.005
In this issue of Structure, Lisa et al. (2015) examine how the PknG protein kinase of M. tuberculosis efficiently binds and phosphorylates substrates. The work highlights interesting parallels between PknG and eukaryotic protein kinases. Tuberculosis is a chronic, single-agent infectious disease that remains one of the greatest killers worldwide (Cole and Alzari, 2007). The disease is caused by Mycobacterium tuberculosis (Mtb), which, being transmitted via aerosols, can survive long term within host cells and evade immune surveillance. Current treatment strategies are limited by the development of multidrug-resistant strains. Therefore, it is essential to improve and further develop drugs and potentially vaccines if we are to avoid reaching a situation that would be like returning to the pre-antibiotic tuberculosis era (Koul et al., 2011). A deeper understanding of the molecular pathways governing pathogenicity and persistence of Mtb is thus of fundamental importance. The work presented by Lisa et al. (2015) unravels the molecular details of the activity and regulation of the protein kinase PknG of Mtb. PknG is a key member of the eukaryotic-like S/T protein kinase family that comprises a total of 11 Mtb kinases. Although most of the family members are membrane proteins, PknG represents one of the two soluble kinases, and it has an important role in Mtb pathogenicity. Although PknG is not essential for the growth of Mtb, it is crucial for Mtb’s survival in the host cell, as it inhibits the phagosome-lysosome fusion, and thus it represents an interesting drug target (Walburger et al., 2004). PknG comprises an N-terminal Rubredoxin (Rdx) domain followed by the kinase domain and a C-terminal tetratricopeptide (TPR) domain (Av-Gay and Everett, 2000). As it has a substitution of the Arg residue preceding the Asp catalytic base, PknG is a non-RD kinase that often do not require phosphorylation of the acti-
vation loop to stabilize the active kinase conformation (Krupa et al., 2004). Instead, PknG possesses four threonine autophosphorylation sites close to the N terminus. These sites are bound by the phospho-threonine binding FHA domain of GarA, the only PknG substrate known to date (O’Hare et al., 2008). In enzymatic assays, Lisa et al. show that the PknG N-terminal region containing the threonine autophosphorylation sites is needed for efficient phosphorylation of full-length GarA, whereas it has no effect on phosphorylation of a short GarA-derived substrate peptide that is phosphorylated much less efficiently than full-length GarA. This mechanism that employs phosphorylation-dependent secondary interactions of kinases with their substrates is also commonly found in eukaryotic kinases to enable the specific and efficient phosphorylation of substrates in cells. The most prominent examples include cytoplasmic tyrosine kinases, which all contain phospho-tyrosine binding SH2 domains, which enhance ‘‘processive’’ multi-site phosphorylation of substrates, as well as many AGC kinases that contain the PIF-binding pocket in their N lobes, which docks to pre-phosphorylated downstream kinases and enables their activation (Arencibia et al., 2013; Jin and Pawson, 2012). In contrast to PknG, both mechanisms additionally entail an allosteric activation of the kinase domains that may have evolved later in evolution, whereas GarA docking to PknG has no effect on intrinsic kinase activity (Lisa et al., 2015). In contrast to the N-terminal region, the Rdx domain, which is located just N-terminal to the kinase domain, inhibits intrinsic kinase activity. To get a deeper
understanding of the regulatory impact of the Rdx domain, Lisa et al. solve crystal structures of a fragment of PknG encompassing Rdx and kinase domain in complex with ADP and in complex with a non-hydrolysable ATP analog. The structures reveal a close association between the Rdx domain and the N-lobe of the kinase being stabilized by several interactions including the electrostatic interaction between E125 and H223. Interestingly, the comparison to a previously published, inhibitor-bound structure reveals marked differences in the position of the Rdx domain supporting a regulatory function of this domain (Figures 1A and 1B) (Scherr et al., 2007). Through binding to the N lobe of the kinase domain, the Rdx domain forms a deep substrate-binding pocket, whereas in the presence of the inhibitor AX20017 a hinge motion in the kinase domain places the Rdx domain in closer proximity with the C lobe of the kinase and restricts the entrance to the kinase active site (Figures 1A and 1B). Interestingly, the position and function of the Rdx domain resembles the position of the FRB domain of the eukaryotic mTOR kinase. Although the FRB domain adopts a different secondary structure and is an insertion to the N lobe, the domain extends the substrate-binding pocket in a similar fashion to Rdx in PknG (Figure 1C). Together with accessory domains interacting with the C lobe, the FRB domain of mTOR forms a V-shaped, deep substrate-binding pocket with restricted access regulating mTOR in a negative manner (Yang et al., 2013). Sequence analysis has previously suggested a close relation between PknG and eukaryotic kinases (Av-Gay and
Structure 23, June 2, 2015 ª2015 Elsevier Ltd All rights reserved 975
Structure
Previews might be a valuable target for anti-tuberculosis drug development. ACKNOWLEDGMENTS
S.R. and O.H. are supported by Swiss National Science Foundation grant 31003A_140913. O.H. holds the ISREC Foundation Chair in Translational Oncology. REFERENCES Arencibia, J.M., Pastor-Flores, D., Bauer, A.F., Schulze, J.O., and Biondi, R.M. (2013). Biochim. Biophys. Acta 1834, 1302–1321. Av-Gay, Y., and Everett, M. (2000). Trends Microbiol. 8, 238–244. Cole, S.T., and Alzari, P.M. (2007). Biochem. Soc. Trans. 35, 1321–1324.
Figure 1. Structure of PknG The structure of the PknG fragment encompassing Rdx and kinase domain bound to ATP-gS presented by Lisa et al. (A; PDB: 4Y12) is compared to PknG structure bound to the inhibitor AX20017 that additionally included the TPR domain (B; PDB: 2PZI) (Scherr et al., 2007) as well as the structure of mTOR kinase (C; PDB: 4JSV) (Yang et al., 2013). Domains are color coded as indicated below the structures.
Everett, 2000). The presented structures of PknG now further strengthen this suggestion, showing that the mode of ATP binding is reminiscent to Aurora A kinase and also that peptide substrate binding involves residues similar to other eukaryotic protein kinases. One very remarkable difference, however, lies in the position of helix aC of the kinase N lobe. In eukaryotes, the positioning of this helix enables the formation of a salt bridge between a glutamate residue and lysine residue within the ATP-binding site that is a hallmark of the kinase active state. In contrast, the corresponding residues of PknG are more than 15 A˚ apart from
each other, implying that an interaction between these two residues is not needed for PknG activity. Surprisingly, but in line with the structural analysis, a point mutation of the glutamate residue did not impair kinase activity (Lisa et al., 2015). In summary, the paper by Lisa et al. provides important molecular details on the regulation of kinase activity and substrate recognition of PknG. The presented results highlight striking similarities to regulatory mechanisms employed by eukaryotic kinases. This study greatly improves our understanding of the mechanisms governing activity of PknG and further supports earlier conclusions that PknG
976 Structure 23, June 2, 2015 ª2015 Elsevier Ltd All rights reserved
Jin, J., and Pawson, T. (2012). Philos. Trans. R. Soc. Lond. B Biol. Sci. 367, 2540–2555. Koul, A., Arnoult, E., Lounis, N., Guillemont, J., and Andries, K. (2011). Nature 469, 483–490. Krupa, A., Preethi, G., and Srinivasan, N. (2004). J. Mol. Biol. 339, 1025–1039. Lisa, M.-N., Gil, M., Andre´-Leroux, G., Barilone, N., Dura´n, R., Biondi, R.M., and Alzari, P.M. (2015). Structure 23, this issue, 1039–1048. O’Hare, H.M., Dura´n, R., Cerven˜ansky, C., Bellinzoni, M., Wehenkel, A.M., Pritsch, O., Obal, G., Baumgartner, J., Vialaret, J., Johnsson, K., and Alzari, P.M. (2008). Mol. Microbiol. 70, 1408–1423. Scherr, N., Honnappa, S., Kunz, G., Mueller, P., Jayachandran, R., Winkler, F., Pieters, J., and Steinmetz, M.O. (2007). Proc. Natl. Acad. Sci. USA 104, 12151–12156. Walburger, A., Koul, A., Ferrari, G., Nguyen, L., Prescianotto-Baschong, C., Huygen, K., Klebl, B., Thompson, C., Bacher, G., and Pieters, J. (2004). Science 304, 1800–1804. Yang, H., Rudge, D.G., Koos, J.D., Vaidialingam, B., Yang, H.J., and Pavletich, N.P. (2013). Nature 497, 217–223.
Previews Kinase Regulation in Mycobacterium tuberculosis: Variations on a Theme Sina Reckel1 and Oliver Hantschel1,* 1Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, E ´ cole Polytechnique Fe´de´rale de Lausanne (EPFL), Lausanne 1015, Switzerland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2015.05.005
In this issue of Structure, Lisa et al. (2015) examine how the PknG protein kinase of M. tuberculosis efficiently binds and phosphorylates substrates. The work highlights interesting parallels between PknG and eukaryotic protein kinases. Tuberculosis is a chronic, single-agent infectious disease that remains one of the greatest killers worldwide (Cole and Alzari, 2007). The disease is caused by Mycobacterium tuberculosis (Mtb), which, being transmitted via aerosols, can survive long term within host cells and evade immune surveillance. Current treatment strategies are limited by the development of multidrug-resistant strains. Therefore, it is essential to improve and further develop drugs and potentially vaccines if we are to avoid reaching a situation that would be like returning to the pre-antibiotic tuberculosis era (Koul et al., 2011). A deeper understanding of the molecular pathways governing pathogenicity and persistence of Mtb is thus of fundamental importance. The work presented by Lisa et al. (2015) unravels the molecular details of the activity and regulation of the protein kinase PknG of Mtb. PknG is a key member of the eukaryotic-like S/T protein kinase family that comprises a total of 11 Mtb kinases. Although most of the family members are membrane proteins, PknG represents one of the two soluble kinases, and it has an important role in Mtb pathogenicity. Although PknG is not essential for the growth of Mtb, it is crucial for Mtb’s survival in the host cell, as it inhibits the phagosome-lysosome fusion, and thus it represents an interesting drug target (Walburger et al., 2004). PknG comprises an N-terminal Rubredoxin (Rdx) domain followed by the kinase domain and a C-terminal tetratricopeptide (TPR) domain (Av-Gay and Everett, 2000). As it has a substitution of the Arg residue preceding the Asp catalytic base, PknG is a non-RD kinase that often do not require phosphorylation of the acti-
vation loop to stabilize the active kinase conformation (Krupa et al., 2004). Instead, PknG possesses four threonine autophosphorylation sites close to the N terminus. These sites are bound by the phospho-threonine binding FHA domain of GarA, the only PknG substrate known to date (O’Hare et al., 2008). In enzymatic assays, Lisa et al. show that the PknG N-terminal region containing the threonine autophosphorylation sites is needed for efficient phosphorylation of full-length GarA, whereas it has no effect on phosphorylation of a short GarA-derived substrate peptide that is phosphorylated much less efficiently than full-length GarA. This mechanism that employs phosphorylation-dependent secondary interactions of kinases with their substrates is also commonly found in eukaryotic kinases to enable the specific and efficient phosphorylation of substrates in cells. The most prominent examples include cytoplasmic tyrosine kinases, which all contain phospho-tyrosine binding SH2 domains, which enhance ‘‘processive’’ multi-site phosphorylation of substrates, as well as many AGC kinases that contain the PIF-binding pocket in their N lobes, which docks to pre-phosphorylated downstream kinases and enables their activation (Arencibia et al., 2013; Jin and Pawson, 2012). In contrast to PknG, both mechanisms additionally entail an allosteric activation of the kinase domains that may have evolved later in evolution, whereas GarA docking to PknG has no effect on intrinsic kinase activity (Lisa et al., 2015). In contrast to the N-terminal region, the Rdx domain, which is located just N-terminal to the kinase domain, inhibits intrinsic kinase activity. To get a deeper
understanding of the regulatory impact of the Rdx domain, Lisa et al. solve crystal structures of a fragment of PknG encompassing Rdx and kinase domain in complex with ADP and in complex with a non-hydrolysable ATP analog. The structures reveal a close association between the Rdx domain and the N-lobe of the kinase being stabilized by several interactions including the electrostatic interaction between E125 and H223. Interestingly, the comparison to a previously published, inhibitor-bound structure reveals marked differences in the position of the Rdx domain supporting a regulatory function of this domain (Figures 1A and 1B) (Scherr et al., 2007). Through binding to the N lobe of the kinase domain, the Rdx domain forms a deep substrate-binding pocket, whereas in the presence of the inhibitor AX20017 a hinge motion in the kinase domain places the Rdx domain in closer proximity with the C lobe of the kinase and restricts the entrance to the kinase active site (Figures 1A and 1B). Interestingly, the position and function of the Rdx domain resembles the position of the FRB domain of the eukaryotic mTOR kinase. Although the FRB domain adopts a different secondary structure and is an insertion to the N lobe, the domain extends the substrate-binding pocket in a similar fashion to Rdx in PknG (Figure 1C). Together with accessory domains interacting with the C lobe, the FRB domain of mTOR forms a V-shaped, deep substrate-binding pocket with restricted access regulating mTOR in a negative manner (Yang et al., 2013). Sequence analysis has previously suggested a close relation between PknG and eukaryotic kinases (Av-Gay and
Structure 23, June 2, 2015 ª2015 Elsevier Ltd All rights reserved 975
Structure
Previews might be a valuable target for anti-tuberculosis drug development. ACKNOWLEDGMENTS
S.R. and O.H. are supported by Swiss National Science Foundation grant 31003A_140913. O.H. holds the ISREC Foundation Chair in Translational Oncology. REFERENCES Arencibia, J.M., Pastor-Flores, D., Bauer, A.F., Schulze, J.O., and Biondi, R.M. (2013). Biochim. Biophys. Acta 1834, 1302–1321. Av-Gay, Y., and Everett, M. (2000). Trends Microbiol. 8, 238–244. Cole, S.T., and Alzari, P.M. (2007). Biochem. Soc. Trans. 35, 1321–1324.
Figure 1. Structure of PknG The structure of the PknG fragment encompassing Rdx and kinase domain bound to ATP-gS presented by Lisa et al. (A; PDB: 4Y12) is compared to PknG structure bound to the inhibitor AX20017 that additionally included the TPR domain (B; PDB: 2PZI) (Scherr et al., 2007) as well as the structure of mTOR kinase (C; PDB: 4JSV) (Yang et al., 2013). Domains are color coded as indicated below the structures.
Everett, 2000). The presented structures of PknG now further strengthen this suggestion, showing that the mode of ATP binding is reminiscent to Aurora A kinase and also that peptide substrate binding involves residues similar to other eukaryotic protein kinases. One very remarkable difference, however, lies in the position of helix aC of the kinase N lobe. In eukaryotes, the positioning of this helix enables the formation of a salt bridge between a glutamate residue and lysine residue within the ATP-binding site that is a hallmark of the kinase active state. In contrast, the corresponding residues of PknG are more than 15 A˚ apart from
each other, implying that an interaction between these two residues is not needed for PknG activity. Surprisingly, but in line with the structural analysis, a point mutation of the glutamate residue did not impair kinase activity (Lisa et al., 2015). In summary, the paper by Lisa et al. provides important molecular details on the regulation of kinase activity and substrate recognition of PknG. The presented results highlight striking similarities to regulatory mechanisms employed by eukaryotic kinases. This study greatly improves our understanding of the mechanisms governing activity of PknG and further supports earlier conclusions that PknG
976 Structure 23, June 2, 2015 ª2015 Elsevier Ltd All rights reserved
Jin, J., and Pawson, T. (2012). Philos. Trans. R. Soc. Lond. B Biol. Sci. 367, 2540–2555. Koul, A., Arnoult, E., Lounis, N., Guillemont, J., and Andries, K. (2011). Nature 469, 483–490. Krupa, A., Preethi, G., and Srinivasan, N. (2004). J. Mol. Biol. 339, 1025–1039. Lisa, M.-N., Gil, M., Andre´-Leroux, G., Barilone, N., Dura´n, R., Biondi, R.M., and Alzari, P.M. (2015). Structure 23, this issue, 1039–1048. O’Hare, H.M., Dura´n, R., Cerven˜ansky, C., Bellinzoni, M., Wehenkel, A.M., Pritsch, O., Obal, G., Baumgartner, J., Vialaret, J., Johnsson, K., and Alzari, P.M. (2008). Mol. Microbiol. 70, 1408–1423. Scherr, N., Honnappa, S., Kunz, G., Mueller, P., Jayachandran, R., Winkler, F., Pieters, J., and Steinmetz, M.O. (2007). Proc. Natl. Acad. Sci. USA 104, 12151–12156. Walburger, A., Koul, A., Ferrari, G., Nguyen, L., Prescianotto-Baschong, C., Huygen, K., Klebl, B., Thompson, C., Bacher, G., and Pieters, J. (2004). Science 304, 1800–1804. Yang, H., Rudge, D.G., Koos, J.D., Vaidialingam, B., Yang, H.J., and Pavletich, N.P. (2013). Nature 497, 217–223.