Key Residues in Mycobacterium tuberculosis Protein Kinase G Play a Role in Regulating Kinase Activity and Survival in the Host
2009; Elsevier BV; Volume: 284; Issue: 40 Linguagem: Inglês
10.1074/jbc.m109.036095
ISSN1083-351X
AutoresDivya Tiwari, Rajnish Kumar Singh, Kasturi Goswami, Sunil Kumar Verma, Balaji Prakash, Vinay Kumar Nandicoori,
Tópico(s)Cancer therapeutics and mechanisms
ResumoProtein kinase G (PknG) in Mycobacterium tuberculosis has been shown to modulate phagosome-lysosome fusion. The protein has three distinct domains, an N-terminal Trx domain, a kinase domain, and a C-terminal TPR domain. The present study extensively analyzes the roles of these domains in regulating PknG kinase activity and function. We find that the kinase domain of PknG by itself is inactive, signifying the importance of the flanking domains. Although the deletion of the Trx domain severely impacts the activity of the protein, the C-terminal region also contributes significantly in regulating the activity of the kinase. Apart from this, PknG kinase activity is dependent on the presence of threonine 309 in the p + 1 loop of the activation segment. Mutating the conserved cysteine residues in the Trx motifs makes PknG refractory to changes in the redox environment. In vitro experiments identify threonine 63 as the major phosphorylation site of the protein. Importantly, we find that this is the only site in the protein that is phosphorylated in vivo. Macrophage infection studies reveal that the first 73 residues, the Trx motifs, and the threonine 63 residue are independently essential for modulating PknG-mediated survival of mycobacteria in its host. We have extended these studies to investigate the role of PknG and PknG mutants in the pathogenesis of mycobacteria in mice. Our results reinforce the findings from the macrophage infection experiments, and for the first time demonstrate that the expression of PknG in non-pathogenic mycobacteria allows the continued existence of these bacteria in host tissues. Protein kinase G (PknG) in Mycobacterium tuberculosis has been shown to modulate phagosome-lysosome fusion. The protein has three distinct domains, an N-terminal Trx domain, a kinase domain, and a C-terminal TPR domain. The present study extensively analyzes the roles of these domains in regulating PknG kinase activity and function. We find that the kinase domain of PknG by itself is inactive, signifying the importance of the flanking domains. Although the deletion of the Trx domain severely impacts the activity of the protein, the C-terminal region also contributes significantly in regulating the activity of the kinase. Apart from this, PknG kinase activity is dependent on the presence of threonine 309 in the p + 1 loop of the activation segment. Mutating the conserved cysteine residues in the Trx motifs makes PknG refractory to changes in the redox environment. In vitro experiments identify threonine 63 as the major phosphorylation site of the protein. Importantly, we find that this is the only site in the protein that is phosphorylated in vivo. Macrophage infection studies reveal that the first 73 residues, the Trx motifs, and the threonine 63 residue are independently essential for modulating PknG-mediated survival of mycobacteria in its host. We have extended these studies to investigate the role of PknG and PknG mutants in the pathogenesis of mycobacteria in mice. Our results reinforce the findings from the macrophage infection experiments, and for the first time demonstrate that the expression of PknG in non-pathogenic mycobacteria allows the continued existence of these bacteria in host tissues. Protein kinases are a diverse class of proteins that have been shown to play a critical role in regulating cellular processes by transmitting extracellular cues/intracellular signals to their downstream substrates by phosphorylation of serine, threonine, or tyrosine residues on the substrates. The Mycobacterium tuberculosis genome encodes for 11 eukaryotic-like serine/threonine protein kinases (1Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry 3rd, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. 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Crystal structures of the kinase domains of PknB, PknE, and PknG as well as the sensor domain of PknD, have been determined, and all of them exhibit the typical two lobed organization observed in eukaryotic serine/threonine kinases. The N-terminal lobe contains the ATP binding site, whereas the C-terminal lobe is involved in rendering an active state and in stabilizing interactions with the substrate (29Ortiz-Lombardía M. Pompeo F. Boitel B. Alzari P.M. J. Biol. Chem. 2003; 278: 13094-13100Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 30Young T.A. Delagoutte B. Endrizzi J.A. Falick A.M. Alber T. Nat. Struct. Biol. 2003; 10: 168-174Crossref PubMed Scopus (196) Google Scholar, 31Gay L.M. Ng H.L. Alber T. J. Mol. Biol. 2006; 360: 409-420Crossref PubMed Scopus (51) Google Scholar, 32Scherr N. Honnappa S. Kunz G. Mueller P. Jayachandran R. Winkler F. Pieters J. Steinmetz M.O. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12151-12156Crossref PubMed Scopus (133) Google Scholar, 33Good M.C. Greenstein A.E. Young T.A. Ng H.L. Alber T. J. Mol. Biol. 2004; 339: 459-469Crossref PubMed Scopus (60) Google Scholar, 34Mieczkowski C. Iavarone A.T. Alber T. EMBO J. 2008; 27: 3186-3197Crossref PubMed Scopus (71) Google Scholar). Protein kinase G (PknG) 3The abbreviations used are: PknGprotein kinase GSTPKserine/threonine protein kinaseMBPmaltose-binding proteinDTTdithiothreitolMbpmyelin basic proteinHPLChigh pressure liquid chromatographyGFPgreen fluorescent proteinCFUcolony forming unit. 3The abbreviations used are: PknGprotein kinase GSTPKserine/threonine protein kinaseMBPmaltose-binding proteinDTTdithiothreitolMbpmyelin basic proteinHPLChigh pressure liquid chromatographyGFPgreen fluorescent proteinCFUcolony forming unit. is closely related to the mammalian protein kinase Cα. Unlike most M. tuberculosis serine/threonine protein kinases (STPKs), it does not have a transmembrane domain and has been shown to be localized to the bacterial cytosol and membrane (35Cowley S. Ko M. Pick N. Chow R. Downing K.J. Gordhan B.G. Betts J.C. Mizrahi V. Smith D.A. Stokes R.W. Av-Gay Y. Mol. Microbiol. 2004; 52: 1691-1702Crossref PubMed Scopus (194) Google Scholar). This 82-kDa protein has a Trx domain at the N terminus, a central kinase domain, and a C-terminal TPR motif. It has been shown to autophosphorylate the kinase and the C-terminal TPR domains (35Cowley S. Ko M. Pick N. Chow R. Downing K.J. Gordhan B.G. Betts J.C. Mizrahi V. Smith D.A. Stokes R.W. Av-Gay Y. Mol. Microbiol. 2004; 52: 1691-1702Crossref PubMed Scopus (194) Google Scholar). The gene encoding PknG is located in an operon with glnH. The inactivation of pknG in M. tuberculosis results in a 3-fold higher accumulation of glutamate and glutamine as well as reduced expression of glutamine synthetase (35Cowley S. Ko M. Pick N. Chow R. Downing K.J. Gordhan B.G. Betts J.C. Mizrahi V. Smith D.A. Stokes R.W. Av-Gay Y. Mol. Microbiol. 2004; 52: 1691-1702Crossref PubMed Scopus (194) Google Scholar). However, another report suggests that the deletion of pknG in Mycobacterium bovis BCG does not affect glutamine uptake or intracellular glutamine concentration (36Nguyen L. Walburger A. Houben E. Koul A. Muller S. Morbitzer M. Klebl B. Ferrari G. Pieters J. J. Bacteriol. 2005; 187: 5852-5856Crossref PubMed Scopus (53) Google Scholar). Walburger et al. (37Walburger A. Koul A. Ferrari G. Nguyen L. Prescianotto-Baschong C. Huygen K. Klebl B. Thompson C. Bacher G. Pieters J. Science. 2004; 304: 1800-1804Crossref PubMed Scopus (441) Google Scholar) analyzed the intracellular trafficking of pknG-deficient M. bovis BCG after macrophage infection. Their data revealed rapid lysosomal transfer of phagocytosed (mutant) mycobacteria. The ortholog of PknG in Mycobacterium smegmatis, a non-pathogenic soil bacterium, is not expressed due to an altered ribosome binding site (8Koul A. Choidas A. Tyagi A.K. Drlica K. Singh Y. Ullrich A. Microbiology. 2001; 147: 2307-2314Crossref PubMed Scopus (90) Google Scholar, 23Houben E.N. Walburger A. Ferrari G. Nguyen L. Thompson C.J. Miess C. Vogel G. Mueller B. Pieters J. Mol. Microbiol. 2009; 72: 41-52Crossref PubMed Scopus (29) Google Scholar). Upon infecting macrophages, wild type M. smegmatis is transferred to lysosomal compartments, whereas M. smegmatis carrying overexpressed PknG is largely present in the non-lysosomal fraction of the cell (37Walburger A. Koul A. Ferrari G. Nguyen L. Prescianotto-Baschong C. Huygen K. Klebl B. Thompson C. Bacher G. Pieters J. Science. 2004; 304: 1800-1804Crossref PubMed Scopus (441) Google Scholar). These results suggest a role for PknG in modulating lysosomal transfer of mycobacteria. PknG kinase activity has been shown to be essential for preventing lysosomal transfer of the phagocytosed mycobacteria. PknG was also shown to be secreted into the cytosol of host macrophages during M. bovis BCG infection (37Walburger A. Koul A. Ferrari G. Nguyen L. Prescianotto-Baschong C. Huygen K. Klebl B. Thompson C. Bacher G. Pieters J. Science. 2004; 304: 1800-1804Crossref PubMed Scopus (441) Google Scholar). Although there is no identifiable N-terminal signal sequence for secretion in PknG, it is known that mycobacterium species have alternate secretory pathways that may aid in secretion of such molecules (38Stanley S.A. Raghavan S. Hwang W.W. Cox J.S. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13001-13006Crossref PubMed Scopus (412) Google Scholar, 39Finlay B.B. Falkow S. Microbiol. Mol. Biol. Rev. 1997; 61: 136-169Crossref PubMed Scopus (1165) Google Scholar). Although the essential role of an active form of PknG in M. tuberculosis infection has been established, there are no reports to date regarding the exact mechanism involved. M. tuberculosis PknG shares about 45% identity and 59% homology with corynebacterial PknG (CgPknG), its ortholog in Corynebacterium glutamicum. CgPknG is not autophosphorylated, but rather, is transphosphorylated by another kinase, CgPknA. This transphosphorylation at the C-terminal domain is essential for the activity of the protein and also the phosphorylation of its downstream substrate (40Fiuza M. Canova M.J. Zanella-Cléon I. Becchi M. Cozzone A.J. Mateos L.M. Kremer L. Gil J.A. Molle V. J. Biol. Chem. 2008; 283: 18099-18112Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, in M. tuberculosis, PknG has been reported to be autophosphorylated in vitro, signifying mechanistic differences in the regulation of PknG and CgPknG. protein kinase G serine/threonine protein kinase maltose-binding protein dithiothreitol myelin basic protein high pressure liquid chromatography green fluorescent protein colony forming unit. protein kinase G serine/threonine protein kinase maltose-binding protein dithiothreitol myelin basic protein high pressure liquid chromatography green fluorescent protein colony forming unit. In this study, we have focused on deciphering the regulatory roles of the various domains of M. tuberculosis PknG. We have utilized GarA as the substrate in in vitro kinase assays, and used GarA phosphorylation as readout of PknG activity. Results of experiments with various deletion mutants of PknG show that although the N-terminal region of PknG is crucial, deleting the C-terminal region also results in loss of activity. In addition, we demonstrate that PknG is more active in an oxidizing environment rather than in a reducing environment and that Trx motifs play a role in sensing the redox environment. A major finding of this investigation is that threonine 63 (Thr-63) is a key phosphorylation site in the N-terminal domain, and is the only site phosphorylated in vivo. Macrophage infection experiments show that the N-terminal region of PknG, including the in vivo phosphorylation site Thr-63 and the Trx motifs are essential for PknG-mediated evasion of lysosomal transfer of mycobacteria in host macrophages. Results from mice infection studies with M. smegmatis expressing PknG or PknG mutants conclusively demonstrate a role for PknG in the persistence of pathogenic mycobacteria in its host. Most importantly, deletion of the N-terminal 73 residues, or mutating the Trx motifs, results in the abrogation of PknG-mediated survival of mycobacteria in host tissues. Restriction/modification enzymes were obtained from New England Biolabs. Cloning and expression vectors pQE2 (Qiagen) and pMAL-c2X (New England Biolabs) were purchased from the respective sources. pVV16 (an Escherichia coli-Mycobacterium shuttle vector) was kindly provided by TB Vaccine Testing and Research Material Contract. [γ-32P]ATP (6000 Ci/mmol) was purchased from PerkinElmer Life Sciences. Inorganic [32P]orthophosphate (H332PO4) was obtained from the Board of Radiation and Isotope Technology, Hyderabad, India. Oligonucleotide primers and analytical grade chemicals were purchased from Sigma. Sequencing of DNA was performed by MWG (India). Sequences of all the primers used and the details of all the plasmid constructs used in this study are given under supplemental Materials. Both pMAL-c2X- and pQE2-based constructs were transformed in DH5α competent cells for protein expression. Fresh transformants were grown in 500–1000 ml of LB medium containing 100 μg/ml ampicillin to a cell density of A600 of ∼0.6. Purification of PknG was carried out as described earlier (37Walburger A. Koul A. Ferrari G. Nguyen L. Prescianotto-Baschong C. Huygen K. Klebl B. Thompson C. Bacher G. Pieters J. Science. 2004; 304: 1800-1804Crossref PubMed Scopus (441) Google Scholar). For GarA purification, cultures were induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside, followed by incubation at 37 °C for 5 h. His-tagged proteins were purified as described (37Walburger A. Koul A. Ferrari G. Nguyen L. Prescianotto-Baschong C. Huygen K. Klebl B. Thompson C. Bacher G. Pieters J. Science. 2004; 304: 1800-1804Crossref PubMed Scopus (441) Google Scholar). Fractions were analyzed on SDS-PAGE and those containing PknG/GarA were pooled and dialyzed against a Tris-based buffer (10 mm Tris-HCl, pH 7.5, 20 mm NaCl, and 20% glycerol). Maltose-binding protein-tagged (MBP) proteins were purified following the manufacturer's recommendations (New England Biolabs). All the purified proteins were estimated and stored at −70 °C until further use. In vitro kinase assays were performed in a 40-μl reaction containing 25 mm HEPES-NaOH, pH 7.4, 20 mm magnesium acetate, 20 μm MnCl2, 1 mm DTT, 200 μm sodium orthovanadate, 100 μm cold ATP, 10 μCi of [γ-32P]ATP, and 5 pmol of GarA or other substrates such as myelin basic protein (Mbp), with or without 1 pmol of PknG or its fragments, for 10 min at 30 °C. The reactions were stopped by adding 15 μl of 4× SDS sample buffer followed by heating at 95 °C for 5 min. Reactions were resolved on 12% SDS-PAGE gels, and either transferred to nitrocellulose membrane or stained with Coomassie/silver and exposed to x-ray films for autoradiography. For quantitation, the desired bands were cut from the gels, soaked in scintillation mixture W (Spectrochem), and counts were taken using a liquid scintillation counter (Packard analyzer Tri-carb 2100 TR). Kinase assays in oxidized versus reduced environments were carried out as described above in the presence of 1 mm oxidized or reduced DTT, respectively. For these experiments, PknG and its mutants were purified in the absence of DTT or β-mercaptoethanol. To determine the kinetic constants, reactions were carried out in a 40-μl volume, in 25 mm HEPES-NaOH, pH 7.4, 20 mm magnesium acetate, and 1 mm DTT, containing 50 μm cold ATP, 10 μCi of [γ-32P]ATP, and 50, 125, or 250 nm PknG, PknG-(1–420), or PknG-(74–750), respectively, for 20 min at 30 °C. Concentrations of GarA in the reaction varied from 0.125 to 5 μm. The concentration (nm) of phosphorylated GarA was determined using the counts per nanomolar ATP. The rate of the phosphotransfer reaction was defined as nanomolar GarA phosphorylated/min/micromolar enzyme. Km and Vmax values were determined by non-linear regression analyses carried out with GraphPad prism software. For two-dimensional phosphopeptide maps or phosphoamino acid analysis, bands corresponding to the protein(s) of interest were excised from the nitrocellulose membrane and digested with mass spectrometry grade trypsin gold (Promega). The peptides were analyzed by two-dimensional resolution on thin-layer cellulose plates (41Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1274) Google Scholar). Aliquots of the tryptic peptide mixtures were further processed and phosphoamino acid analysis was carried out as described (41Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1274) Google Scholar). Reverse phase-HPLC was performed using a C-18 column (PerkinElmer Life Sciences). 20 μg of Radiolabeled PknG was subjected to trypsin digestion and mixed with 200 μg of trypsin-digested PknG. This mixture was resolved by HPLC (PerkinElmer Life Sciences) using a 20–80% acetonitrile gradient. The peaks were monitored with UV as well as the radioactive detector and the peaks that were detected with the radioactive detector were collected. Two-dimensional peptide maps of these collected radioactive peaks were performed as described (41Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1274) Google Scholar). M. smegmatis harboring pVV16-PknG, pVV16-PknG-K181M, or pVV16-PknG-T63A were grown in 200 ml of Middlebrook 7H9 medium (Difco) supplemented with albumin, dextrose, and catalase and 25 μg/ml kanamycin until an O.D. of 1.5 was reached. Cells were harvested by centrifugation at 3,000 × g for 10 min at room temperature and washed once with phosphate-free 7H9 medium, followed by phosphate starvation in 200 ml of 7H9 phosphate-free medium for 6 h. Cells were harvested as described, and resuspended in 50 ml of phosphate-free medium containing phosphatase inhibitors (sodium orthovanadate, β-glycerophosphate, sodium fluoride, and sodium pyrophosphate) and 20 mCi of [32P]orthophosphoric acid and incubated at 37 °C for 8 h at 100 × g. Cells were harvested and lysed using a bead beater in lysis buffer containing phosphate-buffered saline and protease inhibitors. The cell lysates were clarified by centrifugation at 15,000 × g for 60 min, and the supernatant was incubated with nickel-agarose beads overnight at 4 °C. The beads were then thoroughly washed with lysis buffer containing 5 mm imidazole, and resuspended in SDS sample buffer. The samples were resolved on 10% SDS-PAGE, transferred to nitrocellulose membrane, and the radiolabeled bands were visualized by phosphorimaging. M. smegmatis expressing green fluorescent protein (GFP) was transformed with pVV16 vector, PknG, or PknG mutants. Infection experiments were carried out as described (37Walburger A. Koul A. Ferrari G. Nguyen L. Prescianotto-Baschong C. Huygen K. Klebl B. Thompson C. Bacher G. Pieters J. Science. 2004; 304: 1800-1804Crossref PubMed Scopus (441) Google Scholar) except that lysotracker red was used to detect lysosomes. Briefly, J774A.1 cells (5 × 105) were plated on coverslips and infected at a multiplicity of infection of 1:10 for 1 h, followed by a 3-h chase. Coverslips were removed at the given time points and stained with 0.5 μm lysotracker red diluted in Dulbecco's modified Eagle's medium for 10 min in the dark at 37 °C followed by 3 washes with phosphate-buffered saline and fixation with 4% paraformaldehyde. Coverslips were then mounted and visualized using either a Carl Zeiss Imager M1 fluorescence microscope or a Carl Zeiss Axiovision LSM510 meta confocal microscope. C57BL/6J mice were obtained from The Jackson Laboratories and maintained in pathogen-free autoclaved cages in isolators. Three mice per group were injected with 2 × 107 colony forming units (CFU) of M. smegmatis (in 0.9% NaCl) transformed with pVV16 vector, PknG, or PknG mutants by intraperitoneal injection. The final inocula were diluted and plated to confirm the actual bacterial input. The survival of recombinant mycobacteria in mouse organs were determined at 2, 5, and 10 days post-infection as described (42Collins F.M. Tubercle. 1985; 66: 267-276Abstract Full Text PDF PubMed Scopus (38) Google Scholar, 43Dheenadhayalan V. Delogu G. Brennan M.J. Microbes. Infect. 2006; 8: 262-272Crossref PubMed Scopus (109) Google Scholar). Briefly, the mice were sacrificed and the liver, lung, and spleen were removed aseptically, and homogenized using a Down's homogenizer. The tissue homogenates were serially diluted and aliquots from each dilution were plated in triplicate on Middlebrook 7H10 agar plates. Total CFUs obtained were normalized with respect to the bacterial input. A comparative analysis of the proteomes of wild type and the pknG deletion mutant of C. glutamicum led to the identification of OdhI as a likely substrate of CgPknG (44Niebisch A. Kabus A. Schultz C. Weil B. Bott M. J. Biol. Chem. 2006; 281: 12300-12307Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). OdhI is homologous to the mycobacterial protein GarA, a protein known to be secreted in a truncated form into M. tuberculosis culture filtrate (CFP-17), and to induce a high interferon-γ response in inbred mice (45Weldingh K. Rosenkrands I. Jacobsen S. Rasmussen P.B. Elhay M.J. Andersen P. Infect. Immun. 1998; 66: 3492-3500Crossref PubMed Google Scholar). This protein has earlier been identified as a substrate of M. tuberculosis PknB, and the PknB-mediated phosphorylation sites have been determined (46Villarino A. Duran R. Wehenkel A. Fernandez P. England P. Brodin P. Cole S.T. Zimny-Arndt U. Jungblut P.R. Cerveñansky C. Alzari P.M. J. Mol. Biol. 2005; 350: 953-963Crossref PubMed Scopus (128) Google Scholar). As CgPknG and M. tuberculosis PknG (PknG) share a high degree of homology, we investigated the possibility of GarA being a substrate of PknG. Accordingly, the gene encoding PknG was amplified from a BAC library and cloned into pQE2 vector, and the overexpressed protein was purified to near homogeneity (Fig. 1A). Similarly, the gene encoding GarA (Rv1827) was cloned into pQE2, and overexpressed GarA was purified to homogeneity (data not shown). In vitro kinase assays carried out with the purified PknG using GarA as test substrate demonstrated that GarA is a substrate of PknG (Fig. 1B). In an earlier report, PknG was shown to phosphorylate Mbp, the universal kinase substrate (8Koul A. Choidas A. Tyagi A.K. Drlica K. Singh Y. Ullrich A. Microbiology. 2001; 147: 2307-2314Crossref PubMed Scopus (90) Google Scholar). Interestingly, when we compared the ability of PknG to phosphorylate GarA and various canonical kinase substrates, only a weak phosphorylation of histone HIIA and Mbp was detected in comparison to the phosphorylation seen with GarA. As apparent from Fig. 1C, GarA is the optimal substrate among the four that were tested, and hence GarA was used as the substrate to assess PknG kinase activity in further experiments. Phosphoamino acid analysis of phosphorylated PknG showed that phosphorylation was exclusively on threonine residues (Fig. 1D). Among the 11 kinases annotated in the M. tuberculosis genome, PknG is unique due to the presence of an N-terminal extension preceding the kinase domain. In the remaining STPKs, the kinase domain is located in the N terminus followed by regions that, in some of the kinases, regulate their activity (2Av-Gay Y. Everett M. Trends Microbiol. 2000; 8: 238-244Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 47Grundner C. Gay L.M. Alber T. Protein Sci. 2005; 14: 1918-1921Crossref PubMed Scopus (76) Google Scholar, 48Thakur M. Chaba R. Mondal A.K. Chakraborti P.K. J. Biol. Chem. 2008; 283: 8023-8033Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The kinase domain of PknG is located between amino acid residues 151 and 396. The 150-amino acid N-terminal region that precedes it harbors the conserved Trx motifs. The kinase domain is followed by a 354-amino acid C-terminal region showing homology to a TPR domain. To investigate the roles of these flanking regions in modulating PknG kinase activity, we created various deletion mutants of PknG (Fig. 2A). Analysis of proteins on SDS-PAGE showed that all are purified to near homogeneity
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