The Mycobacterium tuberculosis Ser/Thr Kinase Substrate Rv2175c Is a DNA-binding Protein Regulated by Phosphorylation
2009; Elsevier BV; Volume: 284; Issue: 29 Linguagem: Inglês
10.1074/jbc.m109.019653
ISSN1083-351X
AutoresMartin Cohen‐Gonsaud, Philippe Barthe, Marc J. Canova, Charlotte Stagier-Simon, Laurent Kremer, Christian Roumestand, Virginie Molle,
Tópico(s)Biochemical and Molecular Research
ResumoRecent efforts have underlined the role of serine/threonine protein kinases in growth, pathogenesis, and cell wall metabolism in Mycobacterium tuberculosis. Although most kinases have been investigated for their physiological roles, little information is available regarding how serine/threonine protein kinase-dependent phosphorylation regulates the activity of kinase substrates. Herein, we focused on M. tuberculosis Rv2175c, a protein of unknown function, conserved in actinomycetes, and recently identified as a substrate of the PknL kinase. We solved the solution structure of Rv2175c by multidimensional NMR and demonstrated that it possesses an original winged helix-turn-helix motif, indicative of a DNA-binding protein. The DNA-binding activity of Rv2175c was subsequently confirmed by fluorescence anisotropy, as well as in electrophoretic mobility shift assays. Mass spectrometry analyses using a combination of MALDI-TOF and LC-ESI/MS/MS identified Thr9 as the unique phosphoacceptor. This was further supported by complete loss of PknL-dependent phosphorylation of an Rv2175c_T9A mutant. Importantly, the DNA-binding activity was completely abrogated in a Rv2175c_T9D mutant, designed to mimic constitutive phosphorylation, but not in a mutant lacking the first 13 residues. This implies that the function of the N-terminal extension is to provide a phosphoacceptor (Thr9), which, following phosphorylation, negatively regulates the Rv2175c DNA-binding activity. Interestingly, the N-terminal disordered extension, which bears the phosphoacceptor, was found to be restricted to members of the M. tuberculosis complex, thus suggesting the existence of an original mechanism that appears to be unique to the M. tuberculosis complex. Recent efforts have underlined the role of serine/threonine protein kinases in growth, pathogenesis, and cell wall metabolism in Mycobacterium tuberculosis. Although most kinases have been investigated for their physiological roles, little information is available regarding how serine/threonine protein kinase-dependent phosphorylation regulates the activity of kinase substrates. Herein, we focused on M. tuberculosis Rv2175c, a protein of unknown function, conserved in actinomycetes, and recently identified as a substrate of the PknL kinase. We solved the solution structure of Rv2175c by multidimensional NMR and demonstrated that it possesses an original winged helix-turn-helix motif, indicative of a DNA-binding protein. The DNA-binding activity of Rv2175c was subsequently confirmed by fluorescence anisotropy, as well as in electrophoretic mobility shift assays. Mass spectrometry analyses using a combination of MALDI-TOF and LC-ESI/MS/MS identified Thr9 as the unique phosphoacceptor. This was further supported by complete loss of PknL-dependent phosphorylation of an Rv2175c_T9A mutant. Importantly, the DNA-binding activity was completely abrogated in a Rv2175c_T9D mutant, designed to mimic constitutive phosphorylation, but not in a mutant lacking the first 13 residues. This implies that the function of the N-terminal extension is to provide a phosphoacceptor (Thr9), which, following phosphorylation, negatively regulates the Rv2175c DNA-binding activity. Interestingly, the N-terminal disordered extension, which bears the phosphoacceptor, was found to be restricted to members of the M. tuberculosis complex, thus suggesting the existence of an original mechanism that appears to be unique to the M. tuberculosis complex. In response to its environment, Mycobacterium tuberculosis (M. tb) 3The abbreviations used are: M. tbM. tuberculosisEMSAelectrophoretic mobility shift assaySTPKSer/Thr protein kinaseTEVtobacco etch virusHSQCheteronuclear single quantum coherenceHTHhelix-turn-helixwHTHwinged HTHMSmass spectrometry.3The abbreviations used are: M. tbM. tuberculosisEMSAelectrophoretic mobility shift assaySTPKSer/Thr protein kinaseTEVtobacco etch virusHSQCheteronuclear single quantum coherenceHTHhelix-turn-helixwHTHwinged HTHMSmass spectrometry. activates or represses the expression of a number of genes to promptly adjust to new conditions. More precisely, during the infection process, cross-talk of signals between the host and the bacterium take place, resulting in reprogramming the host signaling network. Many of these stimuli are transduced in the bacteria via sensor kinases, enabling the pathogen to adapt its cellular response to survive in hostile environments. Although the two-component systems represent the classic prokaryotic mechanism for detection and response to environmental changes, the serine/threonine and tyrosine protein kinases (STPKs) associated with their phosphatases have emerged as important regulatory systems in prokaryotic cells (1.Stock J.B. Ninfa A.J. Stock A.M. Microbiol. Rev. 1989; 53: 450-490Crossref PubMed Google Scholar, 2.Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3797) Google Scholar, 3.Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2584) Google Scholar). M. tb contains eleven STPKs (4.Cole 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. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6478) Google Scholar, 5.Av-Gay Y. Everett M. Trends Microbiol. 2000; 8: 238-244Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar), and most are being investigated for their physiological roles and potential application for future drug development to combat tuberculosis (6.Wehenkel A. Bellinzoni M. Graña M. Duran R. Villarino A. Fernandez P. Andre-Leroux G. England P. Takiff H. Cerveñansky C. Cole S.T. Alzari P.M. Biochim. Biophys. Acta. 2008; 1784: 193-202Crossref PubMed Scopus (144) Google Scholar). Through phosphorylation these STPKs are also thought to play important functions in cell signaling responses as well as in essential metabolic pathways. The cell wall of M. tb plays a critical role in the defense of this pathogen in the host, and changes in cell wall composition in response to various environmental stimuli are critical to M. tb adaptation during infection. Although little is known regarding the cell wall regulatory mechanisms in M. tb, there is now an increasing body of evidence indicating that these processes largely rely on STPK-dependent mechanisms (7.Sharma K. Gupta M. Krupa A. Srinivasan N. Singh Y. FEBS J. 2006; 273: 2711-2721Crossref PubMed Scopus (65) Google Scholar, 8.Molle V. Brown A.K. Besra G.S. Cozzone A.J. Kremer L. J. Biol. Chem. 2006; 281: 30094-30103Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 9.Veyron-Churlet R. Molle V. Taylor R.C. Brown A.K. Besra G.S. Zanella-Cléon I. Fütterer K. Kremer L. J. Biol. Chem. 2009; 284: 6414-6424Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). M. tuberculosis electrophoretic mobility shift assay Ser/Thr protein kinase tobacco etch virus heteronuclear single quantum coherence helix-turn-helix winged HTH mass spectrometry. M. tuberculosis electrophoretic mobility shift assay Ser/Thr protein kinase tobacco etch virus heteronuclear single quantum coherence helix-turn-helix winged HTH mass spectrometry. Moreover, little information on the range of functions regulated by the STPKs is available, and the complicated mycobacterial phosphoproteome is still far from being deciphered. Understanding mycobacterial kinase biology has been severely impeded by the difficulty to identify direct kinase substrates and the subsequent characterization of the phosphorylation site(s). However, several recent studies have reported the identification and characterization of the phosphorylation sites in substrates related to various metabolic pathways in mycobacteria. These include the Fork Head associated-containing protein GarA, a key regulator of the tricarboxylic cycle (10.O'Hare H.M. Durán R. Cerveñansky C. Bellinzoni M. Wehenkel A.M. Pritsch O. Obal G. Baumgartner J. Vialaret J. Johnsson K. Alzari P.M. Mol. Microbiol. 2008; 70: 1408-1423Crossref PubMed Scopus (130) Google Scholar, 11.Villarino 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); PbpA, a penicillin-binding protein required for cell division (12.Dasgupta A. Datta P. Kundu M. Basu J. Microbiology. 2006; 152: 493-504Crossref PubMed Scopus (132) Google Scholar); Wag31, a homologue of the cell division protein DivIVA that regulates growth, morphology, and polar cell wall biosynthesis in mycobacteria (13.Kang C.M. Nyayapathy S. Lee J.Y. Suh J.W. Husson R.N. Microbiology. 2008; 154: 725-735Crossref PubMed Scopus (187) Google Scholar); the β-ketoacyl acyl carrier protein synthase mtFabH, which participates in mycolic acid biosynthesis (9.Veyron-Churlet R. Molle V. Taylor R.C. Brown A.K. Besra G.S. Zanella-Cléon I. Fütterer K. Kremer L. J. Biol. Chem. 2009; 284: 6414-6424Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar); the anti-anti-sigma factor Rv0516c (14.Greenstein A.E. MacGurn J.A. Baer C.E. Falick A.M. Cox J.S. Alber T. PLoS Pathog. 2007; 3: e49Crossref PubMed Scopus (74) Google Scholar); the alternate sigma factor SigH, which is a central regulator of the response to oxidative stress (15.Park S.T. Kang C.M. Husson R.N. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13105-13110Crossref PubMed Scopus (73) Google Scholar); as well as the essential mycobacterial chaperone GroEL1 (16.Canova M.J. Kremer L. Molle V. J. Bacteriol. 2009; 191: 2876-2883Crossref PubMed Scopus (41) Google Scholar). Therefore, a further characterization of STPKs substrates is critical to unraveling the mechanisms by which STPK-dependent phosphorylation induces modifications, thus regulating their activity, ultimately conditioning biological responses in mycobacteria. Such studies may also provide the key to designing new inhibitors that target signal transduction pathways specific to M. tb. We recently characterized a novel substrate/kinase pair in M. tb, PknL/Rv2175c (17.Canova M.J. Veyron-Churlet R. Zanella-Cleon I. Cohen-Gonsaud M. Cozzone A.J. Becchi M. Kremer L. Molle V. Proteomics. 2008; 8: 521-533Crossref PubMed Scopus (51) Google Scholar). pknL is associated with the ∼30-kb dcw (division cell wall) gene cluster, which encompasses several genes involved in cell wall synthesis and cell division (17.Canova M.J. Veyron-Churlet R. Zanella-Cleon I. Cohen-Gonsaud M. Cozzone A.J. Becchi M. Kremer L. Molle V. Proteomics. 2008; 8: 521-533Crossref PubMed Scopus (51) Google Scholar, 18.Narayan A. Sachdeva P. Sharma K. Saini A.K. Tyagi A.K. Singh Y. Physiol. Genomics. 2007; 29: 66-75Crossref PubMed Scopus (74) Google Scholar), raising the possibility that PknL might participate in the regulation of this gene cluster. Moreover, pknL (Rv2176) is adjacent to the Rv2175c gene, encoding a 16-kDa protein of unknown function. We further demonstrated that phosphorylation of the activation loop Thr-173 residue was required for optimal PknL-mediated phosphorylation of Rv2175c. Moreover, Rv2175c belongs to a mycobacterial "core" of 219 genes, identified by macroarray and bioinformatic analysis, common to M. tb- and Mycobacterium leprae-encoding proteins showing no similarity with proteins from other organisms. The presence of Rv2175c as a member of this set of genes emphasizes the importance of Rv2175c in the physiology of M. tb. In this context, we reasoned that the structural determination of Rv2175c would provide a valuable basis for a better understanding of the function of this protein. Therefore, we have undertaken the structural determination of Rv2175 using multidimensional NMR techniques. Herein, we provide strong evidence that Rv2175c is a DNA-binding protein and investigated how phosphorylation of a unique Thr residue in the N-terminal domain of the protein affects its DNA-binding activity. Strains used for cloning and expression of recombinant proteins were Escherichia coli DH5α (Invitrogen) and E. coli BL21(DE3)Star (Novagen). Strains were grown at 37 °C in LB medium supplemented with 100 μg/ml ampicillin. The Rv2175c gene was amplified by PCR using M. tuberculosis H37Rv genomic DNA as a template and the following primers: #403, 5′-TAT ATA TCG TT CAT atg CCT GGC CGC GCA CCA GGC TCT-3′ (containing an NdeI restriction site underlined) and, #404, 5′-TAT GGA TCC TCA ATA CGC CAT AGC CTG GGC CCG-3′ (containing a BamHI restriction site underlined). This 441-bp amplified product was digested by NdeI and BamHI and ligated into pET15bTev, the variant of pET15b (Novagen) that includes the replacement of the thrombin site coding sequence with a tobacco etch virus (TEV) protease site (19.Cohen-Gonsaud M. Barthe P. Pommier F. Harris R. Driscoll P.C. Keep N.H. Roumestand C. J. Biomol. NMR. 2004; 30: 373-374Crossref PubMed Scopus (12) Google Scholar), and in pETPhos, the variant of pET15bTev that is devoid of putative phosphoacceptors in the His tag fusion (20.Canova M.J. Kremer L. Molle V. Plasmid. 2008; 60: 149-153Crossref PubMed Scopus (30) Google Scholar), thus generating pET15bTev_Rv2175c and pETPhos_Rv2175c, respectively. Site-directed mutagenesis was directly performed on pETPhos_Rv2175c using inverse-PCR amplification with the following self-complementary primers: 5′-GGC CGC GCA CCA GGC TCT GCA CTT GCG CGG GTG GGC AGC-3′ and 5′-GCT GCC CAC CCG CGC AAG TGC AGA GCC TGG TGC GCG GCC-3′ for Rv2175_T9A, 5′-GGC CGC GCA CCA GGC TCT GAC CTT GCG CGG GTG GGC AGC-3′ and 5′-GCT GCC CAC CCG CGC AAG GTC AGA GCC TGG TGC GCG GCC-3′ for Rv2175_T9D, 5′-GGC CGC GCA CCA GGC TCT GAA CTT GCG CGG GTG GGC AGC-3′ and 5′-GCT GCC CAC CCG CGC AAG TTC AGA GCC TGG TGC GCG GCC-3′ for Rv2175_T9E (the corresponding substitutions are shown in bold). The Rv2175c N-terminal truncated fragment lacking the first 13 residues was amplified by PCR using M. tuberculosis H37Rv genomic DNA as a template and the following primers: #615, 5′-TAA TAG CTC ATA TGG GCA GCA TTC CCG CTG GCG ATG AC-3′ (containing an NdeI restriction site underlined) and #404. This 402-bp amplified product was digested by NdeI and BamHI and ligated into pET15bTev thus generating pET15bTev_Rv2175c14–146. All constructs were verified by DNA sequencing. Recombinant E. coli BL21(DE3)Star strains harboring the Rv2175c-expressing constructs pET15bTev_Rv2175c, pETPhos_Rv2175c, pETPhos_Rv2175c_T9A, pETPhos_Rv2175c_T9D, pETPhos_Rv2175c_T9E, and pET15bTev_Rv2175c14–146 were used to inoculate 750 ml of LB medium supplemented with ampicillin and incubated at 37 °C with shaking. When A600 reached 0.6, isopropyl 1-thio-β-d-galactopyranoside was added at a final concentration of 0.2 mm, and growth was continued for an additional 3 h at 37 °C with shaking. Purifications of the His-tagged proteins were performed as reported previously (8.Molle V. Brown A.K. Besra G.S. Cozzone A.J. Kremer L. J. Biol. Chem. 2006; 281: 30094-30103Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 21.Barthe P. Roumestand C. Canova M.J. Kremer L. Hurard C. Molle V. Cohen-Gonsaud M. Structure. 2009; 17: 568-578Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). When required, the protein was treated with TEV protease. Finally, the purified protein was concentrated and applied to a Superdex 75 26/60 (Amersham Biosciences) size exclusion column, equilibrated in buffer 20 mm sodium acetate, pH 4.6, 300 mm NaCl to remove any remaining impurities and the cleaved tag when TEV protease cleavage was performed. The purified Rv2175c protein were identified by SDS-PAGE and stored at −20 °C until required. This protocol was carried out for all the non-labeled constructs and 15N labeled of wild-type and mutant Rv2175c constructs, excepted that the cultures were grown in a minimum media containing 15NH4Cl and 13C6 glucose as the sole nitrogen and carbon sources. The pknL PCR fragment encoding the kinase core domain corresponding to the kinase domain without the juxtamembrane linker of PknL (residues 1–281 out of 399) was amplified by PCR using M. tuberculosis H37Rv genomic DNA as a template and the following primers: #399, 5′-TAT CAT ATG CCG TTG GAG AGC GCG CTG CTG-3′ (containing an NdeI restriction site underlined) and, #400, 5′-TAT GGA TCC TTA CTC CTC GGC GAT CGC CTC CAG-3′ (containing a BamHI restriction site underlined). This 843-bp amplified product was digested by NdeI and BamHI and ligated into pET15bTev. E. coli BL21(DE3)Star cells were transformed with the pET15bTev_pknL1–281 vector expressing the PknL1–281 core domain. Purifications of the His-tagged protein was performed as above (8.Molle V. Brown A.K. Besra G.S. Cozzone A.J. Kremer L. J. Biol. Chem. 2006; 281: 30094-30103Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 21.Barthe P. Roumestand C. Canova M.J. Kremer L. Hurard C. Molle V. Cohen-Gonsaud M. Structure. 2009; 17: 568-578Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). When required, the protein was treated with TEV protease according to the manufacturer's instructions (Invitrogen). NMR experiments were carried out at 14.1 Tesla on a Bruker Avance 600 spectrometer equipped with a 5-mm z-gradient 1H-13C-15N triple resonance cryoprobe. 1H, 13C, and 15N resonances were assigned using standard triple resonance three-dimensional experiments (22.Sattler M. Schleucher J. Griesinger C. Prog. NMR Spectrosc. 1999; 34: 93-158Abstract Full Text Full Text PDF Scopus (1377) Google Scholar) recorded at 30 °C on 0.3 mm15N- or 15N,13C-labeled Rv2175c protein samples dissolved in 10 mm sodium acetate, pH 4.6, 150 mm NaCl with 5% D2O for the lock. 1H chemical shifts were directly referenced to the methyl resonance of di-ethylsilapentane sulfonate, whereas 13C and 15N chemical shifts were referenced indirectly to the absolute frequency ratios 15N/1H = 0.101329118 and 13C/1H = 0.251449530. All NMR spectra were processed with GIFA (23.Pons J.L. Malliavin T.E. Delsuc M.A. J. Biomol. NMR. 1996; 8: 445-452Crossref PubMed Scopus (228) Google Scholar). Nuclear Overhauser effect peaks identified on three-dimensional [1H,15N] and [1H,13C] three-dimensional nuclear Overhauser effect spectroscopy-HSQC were assigned through automated NMR structure calculations with CYANA 2.1 (24.Güntert P. Methods Mol. Biol. 2004; 278: 353-378Crossref PubMed Scopus (1159) Google Scholar). Backbone ϕ and ψ torsion angle constraints were obtained from a data base search procedure on the basis of backbone (15N, HN, 13C′, 13Cα, Hα, and 13Cβ) chemical shifts using the program TALOS (25.Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2732) Google Scholar). Hydrogen bond restraints were derived using standard criteria on the basis of the amide 1H/2H exchange experiments and nuclear Overhauser effect data. When identified, the hydrogen bond was enforced using the following restraints: ranges of 1.8–2.3 Å for d(N-H,O), and 2.7–3.3 Å for d(N,O). The final list of restraints (from which values redundant with the covalent geometry have been eliminated) consisted of 477 intra-residues, 576 sequential, 278 medium-range (1 < i–j ≤ 4), and 345 long range upper bound distance restraints, 190 backbone dihedral angle restraints (ϕ and ϕ), and 66 hydrogen bond restraints. The 30 best structures (based on the final target penalty function values) were minimized with CNS 1.2 according the RECOORD procedure (26.Nederveen A.J. Doreleijers J.F. Vranken W. Miller Z. Spronk C.A. Nabuurs S.B. Güntert P. Livny M. Markley J.L. Nilges M. Ulrich E.L. Kaptein R. Bonvin A.M. Proteins. 2005; 59: 662-672Crossref PubMed Scopus (294) Google Scholar) and analyzed with PROCHECK (27.Laskowski R.A. Moss D.S. Thornton J.M. J. Mol. Biol. 1993; 231: 1049-1067Crossref PubMed Scopus (1078) Google Scholar). The root mean square deviations were calculated with MOLMOL (28.Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6477) Google Scholar). Structural statistics are given in (Table 1).TABLE 1NMR and refinement statistics for Rv2175c protein structuresProteinNMR distance and dihedral constraintsDistance constraintsTotal NOE1676Intra-residue477Inter-residueSequential (|i − j| = 1)576Medium range (|i − j| < 4)278Long-range (|i − j| > 5)345IntermolecularHydrogen bonds66Total dihedral angle restraintsϕ95ψ95χ117Structure statisticsViolations (mean ± S.D.)Maximum distance constraint violation (Å)0.16 ± 0.02Maximum dihedral angle violation (°)4.00 ± 0.88Deviations from idealized geometryBond lengths (Å)0.0095 ± 0.0002Bond angles (°)1.1437 ± 0.0241Impropers (°)1.2872 ± 0.0643Ramachandran plot (%)Most favored region93.2Additionally allowed region6.4Generously allowed region0.3Disallowed region0.1Average pairwise root mean square deviationaThe pairwise root mean square deviation was calculated among 30 refined structures for residues 18–104 and 126–146. (Å)Backbone0.87 ± 0.15Heavy1.54 ± 0.18a The pairwise root mean square deviation was calculated among 30 refined structures for residues 18–104 and 126–146. Open table in a new tab The DNA fragment used in this assay corresponds to 200 bp of non-relevant double stranded M. tuberculosis DNA. The 5′ ends of DNA were labeled using [γ-32P]ATP and T4 polynucleotide kinase. A typical assay mixture contained in 20 μl: 10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 5% (v/v) glycerol, 0.5 mg of bovine serum albumin, radioactive DNA probe (2000 cpm.ml−1), and various amounts of the purified Rv2175c proteins. After 30 min of incubation at room temperature, 20 μl of this mixture was loaded onto a native 4% (w/v) polyacrylamide TBE Ready Gel (Bio-Rad) and electrophoresed in 1% TBE (Tris-Borate-EDTA) buffer for 1 h at 100 V.cm−1. Radioactive species were detected by autoradiography after exposure using direct exposure to films. The 23-bp oligonucleotide with a 3′ reactive amine group (sequence 5′-CTC CAG GTC ACT GTG ACC TCC TC-3′) was purchased from Sigma Genosys. It was covalently labeled with Alexa Fluor 488 succinimidyl ester dye (Invitrogen). A 10-fold molar excess of the dye was added to a solution of DNA in 0.1 m sodium borate, pH 9, buffer, and the reaction was allowed to proceed at room temperature for 3 h with continuous agitation. The reaction was stopped by adding 10% Tris-HCl 1 m. After ethanol precipitation, the labeled oligonucleotide was further purified using reversed-phase chromatography on a C2-C18 column. Finally, it was hybridized with a 1.1/1 molar excess of complementary strand. Steady-state fluorescence anisotropy binding titrations were carried out on a Tecan Saphire II microplate reader, using a 470 nm light emitting diode for excitation, and a monochromator set at 530 nm (bandwith 20 nm) for emission. In vitro phosphorylation of PknL and Rv2175c proteins was carried out for 30 min at 37 °C in a reaction mixture (20 μl) containing buffer P (25 mm Tris-HCl, pH 7.0; 1 mm dithiothreitol; 5 mm MgCl2; 1 mm EDTA) with 200 μCi/ml [γ-32P]ATP. Phosphorylation of Rv2175c by PknL1–281 was performed with 3 μg of Rv2175c in 20 μl of buffer P with 200 μCi/ml [γ-32P]ATP and 500 ng of PknL1–281 for 30 min at 37 °C. The reaction was stopped by addition of an equal volume of 2× sample buffer, and the mixture was heated at 100 °C for 5 min. After electrophoresis, gels were soaked in 16% trichloroacetic acid for 10 min at 90 °C, and dried. Radioactive proteins were visualized by autoradiography using direct exposure to films. In vitro phosphorylation for NMR, EMSAs, and mass spectrometry analysis was performed as described above except that [γ-32P]ATP was replaced by 5 mm non-radioactive ATP and incubated overnight. Purified Rv2175c was subjected to in vitro phosphorylation by PknL1–281 as described above with 5 mm cold ATP. Subsequent mass spectrometry analyses were performed as previously described (29.Fiuza 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). Rv2175c, a 146-amino acid protein with a calculated molecular mass of 16 kDa exhibits very weak similarity to transcriptional regulatory proteins harboring a DNA-binding domain (residues 32–52) with a helix-turn-helix (HTH) motif, as well as homologies to α-helix proteins like cyclines and cytochromes (17.Canova M.J. Veyron-Churlet R. Zanella-Cleon I. Cohen-Gonsaud M. Cozzone A.J. Becchi M. Kremer L. Molle V. Proteomics. 2008; 8: 521-533Crossref PubMed Scopus (51) Google Scholar). Because bioinformatic analyses using several different web-based meta-servers failed to clearly identify Rv2175c as a typical DNA-binding protein, we decided to determine its structure, expecting that it would provide new insights with respect to its function. Rv2175c was isotopically labeled with 15N, and 15N-13C, expressed, and purified from E. coli carrying pET15bTev_Rv2175c. Following removal of its N-terminal His tag using TEV protease, purified Rv2175c was used for the structural determination by multidimensional NMR spectroscopy. By combining the information from the double and triple resonance heteronuclear experiments, we were able to assign all the amide group resonances (except Thr-29) for the non-proline residues (12 prolines), 93.8% of the other backbone resonances (Cα, C′, and Hα), 76.7% of the Cβ resonances, and ∼96% of the side-chain protons. NMR experiments revealed that the N-terminal part (Met1–Ile16) and a large loop (Thr110–Asn122) of Rv2175c were fully disordered. The corresponding residues gave rise to the intense cross-peaks centered at 8.5 ppm in the HSQC spectrum (Fig. 1). Intriguingly, these two regions correspond to the less conserved parts of the protein found in different actinomycetes homologous proteins. Noteworthy, the loop Thr110–Asn122 is present in mycobacteria but missing constantly in Corynebacterium or Streptomyces despite a sequence identity close to 60% for the core residues (Fig. 2). In addition, multiple sequence alignments of Rv2175c homologues revealed that, except for M. tuberculosis and Mycobacterium bovis, most homologues, including Mycobacterium smegmatis, lack the first twelve residues (Fig. 2). One notable exception is Mycobacterium avium paratuberculosis, which carries a 20-amino acid extension with respect to the M. smegmatis sequence.FIGURE 2Multiple sequence alignment of Rv2175c ortholog proteins from corynebacteria and mycobacteria. Sequence alignment of the M. tb Rv2175c homologues in M. bovis, M. avium paratuberculosis, M. marinum, M. leprae, M. avium, M. smegmatis, M. abscessus, Corynebacterium glutamicum, Corynebacterium diphtheriae, and Streptomyces coelicolor. Protein secondary element assignments for Rv2175c are represented on the top of the sequences. Numbering of amino acids corresponds to the Rv2175c protein from M. tuberculosis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We solved the structure of Rv2175c, which corresponds to a monomer constituted of two domains with six α-helices and two β-strands in total (Fig. 3, A and B) with flexibility existing between the N-terminal (residues Pro18 to Val77) and the C-terminal domains (residues Val78 to Tyr146). The 30 best structures were calculated and superimposed for the whole protein heavy atoms (except the first 16 residues) with a root mean square deviation of 1.1 Å, whereas the root mean square deviation calculated for the N-terminal or the C-terminal domain were of 0.7 Å and 0.85 Å, respectively (Fig. 4). Our data indicate that the N-terminal domain, with the topological α1, α2, β1, and β2 order, harbors an unusual prokaryotic winged helix-turn-helix (wHTH) DNA-binding motif missing the typical third helix (30.Aravind L. Anantharaman V. Balaji S. Babu M.M. Iyer L.M. FEMS Microbiol. Rev. 2005; 29: 231-262Crossref PubMed Google Scholar) (Fig. 3, A and B). To our knowledge, the only characterized HTH DNA-binding proteins missing the third helix are the bacteriophage protein from the Xis nucleoprotein filament (31.Sam M.D. Cascio D. Johnson R.C. Clubb R.T. J. Mol. Biol. 2004; 338: 229-240Crossref PubMed Scopus (44) Google Scholar). In the Rv2175c N-terminal domain, the turn following the second strand allows the next six residues (Ile66 and Phe71) to cap the hydrophobic core formed by the two first helices of the wHTH, a role usually devoted to the third helix of the motif (Fig. 3C). Moreover, the Rv2175c C-terminal domain is composed of four anti-parallel helices (α3–α6) and their connecting turns together with a mobile 20-amino acid loop (residues Thr110–Asn122). If most of the hydrophobic residues are buried in the helix bundle, some remain partially solvent-exposed (Leu87, Val138, and Tyr146). Interestingly, in transcriptional regulators harboring a wHTH domain, the C-terminal segment usually possesses a dual role, a functional role as regulatory domain, and a structural role as an actor of protein dimerization when bound to DNA. The relative positioning of these two domains is dictated by contacts between the hydrophobic surface encompassing the residues from the C-terminal α-helices α4 (Ile98, Met99, and Phe103) and α5 residues (Val126 and Leu129) and the N-terminal residues Val22 and Phe70. Overall, the NMR solution structure determination of Rv2175c clearly identified structural features typically found in DNA-binding
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