Loss of kinase activity in Mycobacterium tuberculosis multidomain protein Rv1364c Preeti Sachdeva 1,2 , Azeet Narayan 1 , Richa Misra 1 , Vani Brahmachari 2 and Yogendra Singh 1 1 Institute of Genomics and Integrative Biology (CSIR), Delhi, India 2 Dr B. R. Ambedkar Center for Biomedical Research, University of Delhi, India Despite global efforts to control tuberculosis, it remains an epidemic, with one-third of the world’s population being infected by its etiologic agent, Myco- bacterium tuberculosis, and over 1.5 million people dying from the disease each year. The notorious suc- cess of M. tuberculosis as a highly adapted pathogen rests upon its ability to establish a persistent infection in the hostile environment of the host cell through mechanisms involving transcriptional reprogramming, ensuring metabolic slowdown and upregulation of vir- ulence and stress response pathways [1]. Switching of alternative sigma factors is known to regulate global gene expression to cope with the numerous environ- mental conditions encountered during the establish- ment of a successful infection [2]. The M. tuberculosis genome encodes 13 sigma factors, including 10 alter- Keywords kinase; Mycobacterium; RsbW; Rv1364c; SigF Correspondence Y. Singh, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India Fax: +11 2766 7471 Tel: +11 2766 6156 E-mail: ysingh@igib.res.in (Received 7 September 2008, revised 16 October 2008, accepted 22 October 2008) doi:10.1111/j.1742-4658.2008.06753.x The alternative sigma factors are regulated by a phosphorylation-mediated signal transduction cascade involving anti-sigma factors and anti-anti-sigma factors. The proteins regulating Mycobacterium tuberculosis sigma factor F (SigF), anti-SigF and anti-anti-SigF have been identified, but the factors catalyzing phosphorylation–dephosphorylation have not been well estab- lished. We identified a distinct pathogenic species-specific multidomain pro- tein, Rv1364c, in which the components of the entire signal transduction cascade for SigF regulation appear to be encoded in a single polypeptide. Sequence analysis of M. tuberculosis Rv1364c resulted in the prediction of various domains, namely a phosphatase (RsbU) domain, an anti-SigF (RsbW) domain, and an anti-anti-SigF (RsbV) domain. We report that the RsbU domain of Rv1364c bears all the conserved features of the PP2C- type serine ⁄ threonine phosphatase family, whereas its RsbW domain has certain substitutions and deletions in regions important for ATP binding. Another anti-SigF protein in M. tuberculosis, UsfX (Rv3287c), shows even more unfavorable substitutions in the kinase domain. Biochemical assay with the purified RsbW domain of Rv1364c and UsfX showed the loss of ability of autophosphorylation and phosphotransfer to cognate anti-anti- SigF proteins or artificial substrates. Both the Rv1364c RsbW domain and UsfX protein display very weak binding with fluorescent ATP analogs, despite showing functional interactions characteristic of anti-SigF proteins. In view of conservation of specific interactions with cognate sigma and anti-anti-sigma factor, the loss of kinase activity of Rv1364c and UsfX appears to form a missing link in the phosphorylation-dependent interac- tion involved in SigF regulation in Mycobacterium. Abbreviations GST, glutathione S-transferase; MBP, myelin basic protein; MursiF, multidomain regulator of sigma factor F; PAC, PAS domain-associated C-terminus; PAS domain, Per-Arnt-Sim domain; pNP, p-nitrophenol; pNPP, p-nitrophenyl phosphate; PP, protein phosphatase; SigA, sigma factor A; SigB, sigma factor B; SigF, sigma factor F; TNP-ATP, 2,4,6-trinitrophenyl ATP; UPD, RsbU ⁄ phosphatase domain; VSD, RsbV ⁄ substrate domain; WKD, RsbW ⁄ kinase domain. FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS 6295 native sigma factors [3]. One of the alternative sigma factors of M. tuberculosis, sigma factor F (SigF) is responsible for transcription of gene products of importance to infection and dormancy processes, including genes involved in the biosynthesis of the cell envelope and sigma factor C (SigC) [4]. An M. tuber- culosis SigF-deleted strain grows to a three-fold higher density in stationary phase than the wild-type strain, and is attenuated for virulence in a mouse model of infection [5]. The regulation of expression and activity of sigma factors is brought about by phosphorylation and pro- tein–protein interaction events in a partner-switching mechanism involving anti-sigma factors and anti-anti- sigma factors. M. tuberculosis SigF is related to sigma factor B (SigB), a stress-response specific sigma factor of Bacillus subtilis, and SigF of B. subtilis, a sporula- tion-specific sigma factor [6]. In both the SigB and SigF regulatory pathways of B. subtilis, the activity of the sigma factor is negatively regulated by the cognate anti-sigma factor, RsbW, and SpoIIAB, respectively, which hold the transcription factor in an inactive com- plex. Release of SigB and SigF from the complex is mediated by the anti-anti-sigma factors, RsbV and SpoIIAA, respectively. The action of the anti-anti- sigma factors is counteracted by the kinase activity of the dual-function RsbW and SpoIIAB proteins, which phosphorylate and thereby inactivate their respective anti-anti-sigma factors. Finally, the phosphorylated RsbV and SpoIIAA proteins are reactivated by the action of phosphatases, RsbU and SpoIIE respectively. Other proteins, such as RsbT, act upstream by binding and activating the phosphatase activity of RsbU in B. subtilis [7–9]. Similarly, the M. tuberculosis genome has been shown to possess a bona fide anti-SigF protein, UsfX ⁄ RsbW, as well as two anti-anti-SigF proteins, RsfA and RsfB. RsfB is regulated by phos- phorylation, as a mutation that is believed to mimic phosphorylation renders it nonfunctional. However, RsfB is not phosphorylated by UsfX in an in vitro kinase reaction [10]. The upstream molecules in this event, i.e. a kinase and a phosphatase regulating UsfX–RsfB interaction, remain to be elucidated. The M. tuberculosis genome analysis reveals another potential SigF regulatory gene, Rv1364c encoding a protein with multidomain architecture, in which the entire regulatory cascade akin to the RsbU–RsbW– RsbV system in B. subtilis appears to be present within a single polypeptide with an additional sensor domain, the Per-Arnt-Sim (PAS) domain [11]. Rv1364c is upregulated during nutrient starvation in M. tuber- culosis [12], whereas its Mycobacterium bovis ortholog is upregulated in response to environmental changes encountered within the macrophages [11]. In a yeast two-hybrid-based study, Rv1364c has been reported to interact with SigF as well as UsfX, and interdomain interactions between its RsbW and RsbV domains also occur [13]. The role and mechanism of regulation of the multidomain protein Rv1364c are intriguing, and underline the need to study this component of the M. tuberculosis SigF regulation network. The present study focuses on the functional characterization and role of Rv1364c in phosphorylation–dephosphorylation of anti-anti-sigma factors, which are known to be important for regulation of SigF in M. tuberculosis. Results and Discussion Domain and genomic organization of Rv1364c orthologs The gene product encoded by M. tuberculosis Rv1364c, annotated as rsbU [3], represents a multidomain pro- tein comprising fused units that occur as independent stand-alone proteins in the same and other distant bac- terial species. As the Rv1364c domain composition represents a previously unexemplified unique fusion of the sensor–RsbU–RsbV–RsbW module for SigF regu- lation, we have renamed the protein as putative multi- domain regulator of SigF (MursiF) (Fig. 1A). We performed a comparative genomic analysis of sequenced mycobacterial genomes to identify MursiF orthologs across the Mycobacterium genus, using full- length M. tuberculosis H 37 Rv MursiF as well as its individual domains as query sequences. The analysis revealed the conservation of MursiF orthologs across all sequenced M. tuberculosis strains, namely M. tuber- culosis H 37 Ra, M. tuberculosis F11, M. tuberculosis CDC1551, M. tuberculosis C and all other sequenced slow-growing pathogenic Mycobacterium spp., namely M. bovis AF2122 ⁄ 97, Mycobacterium avium 104, M. avium paratuberculosis K-10, Mycobacterium mari- num and Mycobacterium ulcerans (Fig. 1A). MursiF ortholog is, however, absent in Mycobacterium leprae, where the gene encoding SigF itself is known to be a pseudogene [14]. On the other hand, nonpathogenic fast-growing Mycobacterium spp., such as Mycobacterium smegma- tis, Mycobacterium gilvum, Mycobacterium vanbaalenii, Mycobacterium sp. MCS, Mycobacterium sp. KMS, and Mycobacterium sp. JLS, were found to have no sequence homologous to the complete multidomain module of MursiF. Search of the M. smegmatis gen- ome using the RsbU domain of MursiF as a query sequence revealed a protein annotated as response reg- ulator receiver protein (MSMEG_6131). This protein Functional characterization of Rv1364c P. Sachdeva et al. 6296 FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS comprises two domains, a phosphatase domain with 44% similarity to the MursiF RsbU domain, and a receiver domain analogous to the phosphoacceptor protein of histidine kinases (Fig. 1B). Unlike M. tuber- culosis mursiF, the MSMEG_6131 gene possibly forms an operon (intergenic distance 3 bp) with a gene pair comprising a two-component system sensor kinase (MSMEG_6130) and a response regulator (MSMEG_6128) (Fig. 1B). Interestingly, adjacent and in opposite orientation to this putative operon in M. smegmatis, we identified two genes in tandem, MSMEG_6129 and MSMEG_6127 encoding protein sequences with 44 and 58% similarity to the RsbW domain and the RsbV domain of M. tuberculosis MursiF, respectively (Fig. 1B). The analysis of genomes of other nonpathogenic Mycobacterium spp. namely M. gilvum, M. vanbaalenii, Mycobacterium sp. MCS, Mycobacterium sp. KMS, and Mycobacterium sp. JLS, also revealed the conservation of sequences ortholo- gous to the M. smegmatis response regulator receiver protein (MSMEG_6131) and two-component system (MSMEG_6130, MSMEG_6128) (Fig. 1B). The genes encoding proteins homologous to the RsbW and RsbV domains of MursiF (Fig. 1A) in these species were, however, found to be located far apart from response regulator receiver protein orthologs. The rsbW gene is spaced precisely 25 genes away in the closely related strains Mycobacterium sp. MCS, Mycobacterium sp. KMS, and Mycobacterium sp. JLS, and 15 genes away in the closely related species M. gilvum and M. vanba- alenii, from the gene encoding the response regulator receiver protein (Fig. 1B). These observations, very interestingly, suggest that stand-alone RsbU, RsbW and RsbV encoding genes in nonpathogenic mycobac- terial species have converged to form a gene encoding fused mutidomain RsbU–W–V module, specifically in pathogenic Mycobacterium spp. The selective advan- tage of domain fusion lies in the increased efficiency of coupling and coregulation of the corresponding bio- chemical reaction or signal transduction step as well as expression of the fused domains [15]. It is likely that radically fused genes, which may emerge as a result of Membrane protein Rv1364c RsfA Mb1399 Membrane protein RsfA MAV_1619 Acyl-CoA dehydrogenase Hypothetical protein MAP2361 Hypothetical protein Acyl-CoA dehydrogenase Acyl-CoA dehydrogenase PE-PGRS family pseudogene MUL_3853 Acyl-CoA dehydrogenase PE family protein MM3991 M. bovis M. tuberculosis H 37 Rv M. marinum M. avium paratuberculosis M. ulcerans M. avium M. smegmatis M. sp. KMS M. gilvum M. sp. MCS M. sp. JLS M. vanabaaleni i MSMEG_6131 Glucarate dehydratase Sensor Kinase Response regulator RsbW RsbV PAS domain RsbU /Phosphatase d omain (UPD) RsbW/K inase d omain (WKD) RsbV/Substrate d omain (VSD) Coiled coil AB domain 1 135 165 404 544 653 RsbU/Phosphatase domain 26 140 207 402 1 Hypothetical protein Response regulator Sensor Kinase Mmcs_2688 Hypothetical protein RsbW RsbV Response regulator Sensor Kinase Acyl- transferase Mflv_3268 Phospho- diesterase RsbW RsbV Response regulator Sensor Kinase Acyl- transferase Mvan_2987 Phospho- diesterase RsbW RsbV Hypothetical protein Response regulator Sensor Kinase Mjls_2718 Hypothetical protein RsbW RsbV Response regulator Sensor Kinase Hypothetical protein Mkms_2732 Hypothetical protein RsbW RsbV Signal receiver domain Fig. 1. Schematic representation of domain architecture and genomic organization of Rv1364c and its orthologs in pathogenic Mycobacte- rium spp. (A) and response regulator receiver protein orthologs in nonpathogenic Mycobacterium spp. (B). M. tuberculosis Rv1364c and its orthologs are shown as ( ) arrows, and M. smegmatis response regulator receiver protein and its orthologs are shown as ( ) arrows. The numbers below the domain architecture diagram (shown as boxes) refer to amino acids defining boundaries of each of the domains. The two proteins share a common domain, RsbU ⁄ phosphatase domain ( ). The sequences homologous to other two domains, the RsbW domain ( ) and the RsbV domain ( ), of Rv1364c (A) exist as independent genes in nonpathogenic Mycobacterium spp. (B). represents the region containing a large number of genes separating the response regulator receiver protein and RsbW genes; 25 genes in Mycobacterium sp. MCS, Mycobacterium sp. KMS and Mycobacterium sp. JLS, and 15 genes in M. gilvum and M. vanbaalenii . P. Sachdeva et al. Functional characterization of Rv1364c FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS 6297 genome rearrangements, fix in a population because they have a novel function that is advantageous to the organism [16]. Fusion of domains associated with a reg- ulatory pathway for stress adaptation to form a contig- uous polypeptide may be advantageous in the evolutionary optimization of the genome of pathogenic Mycobacterium spp. The unique pathogen-specific domain architecture and its upregulation in Mycobacte- rium residing in a macrophage environment [11] makes MursiF a protein of utmost importance. Sequence analysis of MursiF In silico analysis was performed to determine the nat- ure and domain features of M. tuberculosis H 37 Rv MursiF. This 653 amino acid protein has an estimated molecular mass of 69.523 Da and a pI value of 4.763. MursiF is predicted to be a soluble protein by analysis programs. MursiF has a PAS sensor domain at its N-terminus, with an adjacent PAC (PAS domain-asso- ciated C-terminus) region (Fig. 1A). PAS domains are sensory modules that undergo conformational changes in the presence of various physical stimuli and ligand molecules [17]. PAS domains show conservation in three-dimensional fold and dynamic properties rather than in amino acid sequences [18]. The MursiF PAS domain has 38% similarity to that of B. subtilis RsbP, an energy stress-dependent serine phosphatase [19]. Interestingly, in addition to a PAS sensor domain and RsbU–W–V module, MursiF has a coiled coil motif spanning residues 135–165, as determined by the multicoil [20] and smart [21] programs (Fig. 1A). The coiled coil motif is a structural motif involved in oligomerization [22], and may play an important role in protein–protein interactions [23], which form the fundamental basis of the SigF regulation mechanism. The RsbU ⁄ phosphatase domain (UPD) of MursiF is homologous to protein phosphatase PP2C-class ser- ine ⁄ threonine phosphatase, and shows 43% similarity with B. subtilis RsbU. Multiple sequence alignment of MursiF and other similar bacterial PP2C family mem- bers shows the conservation of all critical residues of 11 characteristic motifs, including those predicted to be involved in divalent metal ion coordination, Asp211 (subdomain II) and Asp328 (subdomain VIII) (Fig. 2). However, the sequence lacks the Va- and Vb-boxes of the PP2C-type catalytic domain, as reported for the RsbU, RsbX and SpoIIE family of phosphatases [24]. MursiF UPD possesses a PAS sensor domain at its N-terminus (Fig. 1A) in place of a motif for interaction with an activator, RsbT, that is present in Bacillus and Staphylococcus homologs. During environmental stress, RsbT positively regulates RsbU phosphatase activity [25]. No sequences homologous to genes encoding the RsbRST module are present in the M. tuberculosis genome. In this scenario, recruitment of a signaling domain, together with loss of a domain that mediates stress-induced interaction with an activator, suggests a direct sensing mechanism for stress signals by MursiF. The putative anti-SigF domain of MursiF, the RsbW ⁄ kinase domain (WKD), shows 40% similarity to B. subtilis RsbW, a serine kinase belonging to the GHKL family of kinases. This family of kinases is defined by the presence of an ATP-binding fold called the ‘Bergerat fold’, comprising three motifs, the N-, G1- and G2-boxes, which have been found to be conserved in histidine kinases and ATPases [26]. Anal- ysis of the MursiF WKD sequence revealed conserva- tion of most of the residues characteristic of the N-, G1- and G2-boxes; however, some significant vari- ations were observed. Careful comparative analysis of MursiF WKD sequences across all sequenced Myco- bacterium spp. and functionally characterized RsbW proteins from other genera was therefore carried out using multiple sequence alignment (Fig. 3A). We noted that a region speculated to be a part of the ATP lid in functional RsbW sequences of other genera is deleted in the WKD sequences of all MursiF orthologs identi- fied across Mycobacterium spp. (Fig. 3A). The ATP lid changes its conformation on nucleotide binding, and is presumed to couple ATP binding to function-specific interdomain associations [27]. Mutagenesis of the pro- posed hinge of the ATP lid in a GHKL family kinase, EnvZ, has shown that this region is essential for kinase activity [28]. Furthermore, a conserved lysine residue close to the N-box of histidine kinases, which has been shown to be important for nucleotide binding as well as catalysis [29], is substituted in the WKD sequences of all MursiF orthologs (Fig. 3A). The novel genes formed as a result of fusion of two or more genes are believed to experience a burst of rapid adaptive substi- tution shortly after they are formed, followed by a slowing of evolution, which is consistent with increased evolutionary constraint [16]. Less than 50% similarity of MursiF UPD and WKD and the aforementioned divergence of MursiF WKD sequences from their orthologous sequences in other bacteria may be attrib- uted to adaptive evolution. The M. tuberculosis genome encodes another func- tional anti-SigF protein, UsfX (Rv3287c) [10], which has insignificant similarity to the MursiF WKD sequence. UsfX has been shown to catalyze phospho- transfer to an artificial substrate, myelin basic protein (MBP) [30], but not to any of the anti-anti-sigma fac- tor proteins [10,30]. We carried out a comparative analysis of UsfX sequences across all sequenced Myco- Functional characterization of Rv1364c P. Sachdeva et al. 6298 FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS bacterium spp. and functionally characterized RsbW proteins from other genera (Fig. 3B). We found that M. tuberculosis UsfX, surprisingly, has substantially divergent motifs (45% similarity to B. subtilis RsbW), and completely lacks the G1-box consensus sequence (Fig. 3B). Furthermore, the conserved amino acids in the signature sequences of the N- and G2-boxes have been substituted with other less similar residues (Fig. 3B), and the possibility of functional competence of these substitutions remains to be studied. Impor- tantly, a Bordetella BtrW (RsbW ortholog) N-box mutant has been reported to be incapable of phosphor- ylating its substrate, BtrV (RsbV homolog), whereas its BtrW G1-box mutant phosphorylated BtrV to a les- ser extent than its wild-type counterpart and is also defective in the ability to form a stable complex with the phosphorylatable form of BtrV [31]. Similar to MursiF WKD, the UsfX sequence also shows a dele- tion of the ATP lid region as well as an absence of the lysine residue close to the N-box of histidine kinases (Fig. 3A,B). However, in view of the presence of sev- eral divergent motifs in the UsfX sequence, it seems to have acquired a large number of deleterious mutations in an independent evolutionary event. In view of the Fig. 2. Comparison of the MursiF RsbU ⁄ phosphatase domain with PP2C family serine ⁄ threonine phosphatases of similar classes from other organisms. MursiF of M. tuberculosis was aligned with the PP2C domains of RsbU of B. subtilis, SpoIIE of B. subtilis and IcfG of Synecho- cystis sp. using T-COFFEE. Identical amino acids are indicated by asterisks, high similarity is indicated by double dots, and lower similarity is indicated by single dots. The gaps are introduced to optimize the alignment and are indicated by the dashes. Various motifs described in the text are marked and shown in bold. Two conserved aspartate residues, Asp211 and Asp328, involved in binding divalent cations and mutated in the study are indicated as shaded residues. P. Sachdeva et al. Functional characterization of Rv1364c FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS 6299 A B Fig. 3. Comparison of mycobacterial MursiF WKD sequences (A) and mycobacterial UsfX sequences (B) with functionally characterized RsbW sequences from other genera. Alignments were done using T-COFFEE. Identical amino acids are indicated by asterisks, high similarity is indicated by double dots, and lower similarity is indicated by single dots. The gaps are introduced to optimize the alignment and are indicated by the dashes. Various motifs described in the text are marked and shown in bold. Functional characterization of Rv1364c P. Sachdeva et al. 6300 FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS aforementioned deletions and mutations, the ability of MursiF WKD and UsfX protein to act as functional kinases in the SigF regulation cascade is questionable and needs to be addressed. The putative anti-anti-SigF domain of MursiF, the RsbV ⁄ substrate domain (VSD), has 50% similarity to B. subtilis RsbV. The domain has been shown to inter- act with MursiF WKD [13], and has all the conserved features of the anti-anti-sigma factor sequence (Fig. S1). A serine residue, Ser600, at the predicted phosphorylation site is conserved in MursiF VSD (Fig. S1). Protein expression and purification The above-mentioned in silico analysis necessitates bio- chemical analysis of MursiF and UsfX. In order to functionally characterize full-length MursiF as well as each of its domains, the full-length M. tuberculosis H 37 Rv mursiF gene and its individual domains, namely upd, wkd, and vsd, were cloned and expressed as His ⁄ glutathione S-transferase (GST)-tagged recombi- nant proteins in Escherichia coli (Fig. 1A, Table 1). In addition, His-tagged M. tuberculosis SigF and His- tagged and GST-tagged M. tuberculosis UsfX were also overexpressed in E. coli. All the proteins were purified as hexa-His (H) or GST-fusion (G) proteins (H-MursiF, H-UPD, H-WKD, G-WKD, H-VSD, G-VSD, H-UsfX, G-UsfX, SigF) using affinity chro- matography. MursiF and its phosphatase domain (UPD) mutant proteins carrying aspartate residue (D211A, D328A) mutations (H-MursiF-D211A, H-UPD-D211A, H-MursiF-D328A, H-UPD-D328A) were subsequently purified using the same strategy. On electrophoretic analysis of the purified proteins, H-MursiF (and its mutants), H-UPD (and its mutants), H-WKD, G-WKD, H-VSD, G-VSD, H-UsfX and G-UsfX were detected as 73, 47, 18, 44, 14, 37, 22 and 45 kDa proteins, respectively (Fig. 4). These protein sizes were consistent with predicted mole- cular masses along with appropriate hexa-His (3 kDa) or GST (26 kDa) affinity tags, except for UsfX, which migrated at a size slightly higher than the expected molecular mass. The identity of purified proteins was also confirmed by western blot using monoclonal anti- bodies against penta-His and GST (data not shown). MursiF UPD characterization The phosphatase activity of purified full-length MursiF (H-MursiF) and MursiF UPD was determined by its ability to dephosphorylate a small molecule substrate, p-nitrophenyl phosphate (pNPP), thereby forming a p-nitrophenolate ion, which is detected by measuring absorbance at 405 nm (Fig. 5A). Further characteriza- tion revealed that MursiF phosphatase activity has a pH optima of 8.5 and an optimum temperature of 37 °C (data not shown). The activity of H-MursiF was found to be strictly Mn 2+ -dependent (Fig. 5B), being maximal at 2.5 mm Mn 2+ (data not shown). Other cations such as Ca 2+ ,Ba 2+ ,Zn 2+ ,Sr 2+ ,Co 2+ and Ni 2+ failed to substitute for Mn 2+ , whereas Mg 2+ was found to inhibit pNPP hydrolysis by H-MursiF (Fig. 5B). Two conserved aspartate residues, Asp211 and Asp328, at positions known to be involved in metal ion coordination were mutated (Fig. 2). The two Table 1. Summary of expression vectors used in the study. Expression vector Description of protein expressed Name Amino acid Reference pHTc-mursiF Full-length His-tagged MursiF H-MursiF – This study pHTc-mursiFD211A H-MursiF carrying D211A mutation H-MursiF-D211A – This study pHTc-mursiFD328A H-MursiF carrying D328A mutation H-MursiF-D328A – This study pHTc-upd His-tagged MursiF RsbU ⁄ phosphatase domain H-UPD 1–404 This study pHTc-updD211A H-UPD carrying D211A mutation H-UPD-D211A 1–404 This study pHTc-updD328A H-UPD carrying D328A mutation H-UPD-D328A 1–404 This study pHTc-wkd His-tagged MursiF RsbW ⁄ kinase domain H-WKD 404–544 This study pGEX-wkd GST-tagged MursiF WKD G-WKD 404–544 This study pHTc-vsd His-tagged MursiF RsbV ⁄ substrate domain H-VSD 545–653 This study pGEX-vsd GST-tagged MursiF VSD G-VSD 545–653 This study pHTc-usfX His-tagged UsfX H-UsfX – This study pGEX-usfX GST-tagged UsfX G-UsfX – This study pGEX-rsfB GST-tagged RsfB G-RsfB – This study pGEX-Rv2638 GST-tagged Rv2638 G-Rv2638 – This study pLCD1 His-tagged SigF H-SF – [6] pHTc-sigA His-tagged SigA H-SA – This study pGEX-pknB GST-tagged PknB G-PknB – [37] P. Sachdeva et al. Functional characterization of Rv1364c FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS 6301 mutant proteins, H-MursiF-D211A and H-MursiF- D328A, showed  10-fold lower activity than wild- type MursiF (Fig. 6). This further emphasizes the divalent cation dependence of MursiF activity and the crucial role of Asp211 and Asp328 in Mn 2+ coordina- tion. In an attempt to assign MursiF to a specific class of phosphatases, the effect of various class-specific phosphatase inhibitors, such as sodium orthovanadate, calyculin A, cyclosporine, and okadaic acid, was stud- ied. Insensitivity to okadaic acid, a potent inhibitor of the PP2A and PP2B family of phosphatases, is one of the unique characteristics of the PP2C subfamily [32]. None of the PP1, PP2A or PP2B class-specific inhibi- tors, including okadaic acid, had any effect on the phosphatase activity of MursiF. Only sodium fluoride, a nonspecific phosphatase inhibitor, inhibited pNPP hydrolysis by H-MursiF by 30% (Fig. 7). MursiF is therefore an Mn 2+ -dependent PP2C-type alkaline phosphatase that is most active in the physiological temperature range. The purified phosphatase domain (H-UPD) of MursiF was also studied, and was found to have similar characteristics (data not shown). P ro t ei n m ar k er 220 116 97 66 31 21 14.4 220 116 97 66 31 21 45 H- M u rs i F H- M u rs i F D2 1 1A H- M u rs i F D3 2 8A H - UP D H- WKD G - WKD G - V S D H- V S D P ro t ei n m ar k er H- Us f X G - Us f X H- S F Fig. 4. Analysis of recombinant proteins by SDS ⁄ PAGE. Affinity-purified His-tagged (H-) or GST-tagged (G-) fusion proteins were subjected to 13% SDS ⁄ PAGE and stained with Coomassie brilliant blue. The numbers on the left indicate sizes of bands of molec- ular mass marker. The proteins analyzed are indicated at the top of each lane, and details are listed in Table 1. 0 2 4 6 8 A B 0 20 40 60 80 100 120 Time (min) µM pNP formed/µg of protein 0 0.2 0.4 0.6 0.8 1 1.2 1.4 No ion Mg Mn Ca Ba Ni Zn Co Sr Divalent cation ( 5 mM ) A 405 Fig. 5. Biochemical characterization of MursiF phosphatase activity. (A) Time kinetics of phosphatase activity of MursiF were deter- mined by the hydrolysis of a low molecular weight substrate, pNPP, as measured by absorbance at 405 nm. The activity was expressed as micromoles of pNP liberated ⁄ lg of protein. (B) Puri- fied H-MursiF (5 lg) was incubated with pNPP in reaction buffer containing different divalent cations (Mg 2+ ,Ca 2+ ,Ba 2+ ,Zn 2+ ,Sr 2+ , Co 2+ , and Ni 2+ ). A 405 nm for each of the reactions at 100 min is plot- ted. Each value is the average of three individual reactions and is given as mean ± SD. 0 0.2 0.4 0.6 0.8 1 1.2 MursiF- WT MursiF- D211A MursiF- D328A Relative A 405 Fig. 6. Comparative analysis of phosphatase activities of wild-type and mutant MursiF. MursiF-WT, unaltered ⁄ wild-type H-MursiF; MursiFD211A, H-MursiF carrying the D211A mutation; Murs- iFD328A, H-MursiF carrying the D328A mutation (Table 1). Con- served aspartate residues (as marked in Fig. 2), predicted to be involved in metal ion coordination, were mutated, and proteins were assayed for pNPP hydrolysis. A 405 nm for wild-type protein is normalized as 1, and relative values for mutant proteins are plotted. Each value is the average of three individual reactions and is given as mean ± SD. Functional characterization of Rv1364c P. Sachdeva et al. 6302 FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS To study protein dephosphorylation by MursiF, [ 32 P]serine ⁄ threonine-phosphorylated or [ 32 P]tyrosine- phosphorylated artificial substrates such as casein, histone and MBP were incubated with H-MursiF and H-UPD, but no dephosphorylation of any of the artifi- cial substrates was seen. The reaction conditions were varied over a large number of biophysical and bio- chemical parameters. In all cases, the level of free inor- ganic phosphate released from 32 P-phosphorylated substrates on incubation with MursiF was found to be insignificant and almost comparable to that seen in the presence of H-MursiF-D211A or H-MursiF-D328A (data not shown). In this regard, MursiF behaves dif- ferently from other PP2C-type phosphatases, but its refractory behavior to artificial substrates is in agree- ment with that reported for the RsbU homolog in Synechocystis sp. IcfG (Slr1860) [33]. Similarly, Bacillus RsbU and RsbX protein phos- phatases display strict specificities for a single homolo- gous phosphoprotein, RsbV and RsbS, respectively [34]. Another member, SpoIIE, does not dephosphory- late its physiological substrate protein, SpoIIAA, following replacement of the phospho-serine residue at its phosphorylation site by a phospho-threonine [35]. The lack of Va- and Vb-boxes in the PP2C-type cata- lytic domain is a feature shared by MursiF UPD with RsbU, RsbX and SpoIIE phosphatases, which are known to be divergent PP2C-type phosphatases [24] (Fig. 2). The residues in the Va- and Vb-boxes of the PP2C phosphatase catalytic domain have not been characterized for their role in catalysis, but their selec- tive absence in RsbU-like phosphatases may possibly be relevant for the stringent specificity of this group of phosphatases. MursiF WKD characterization To examine whether MursiF retains kinase activity in spite of the loss of critical residues involved in ATP binding, as observed in our in silico analysis of MursiF WKD (Fig. 3A), purified H-MursiF as well as the H-WKD and G-WKD proteins were incubated with [ 32 P]ATP[cP] in appropriate buffer conditions. No autophosphorylation signal was observed for any of the proteins (data not shown). As expected, H-MursiF or domain alone (H-WKD and G-WKD) did not phosphorylate its purified RsbV ⁄ substrate domain (H- VSD, G-VSD), despite the presence of the conserved phosphorylation site Ser600 in VSD (Fig. S1). Also, no phosphotransfer was seen on the artificial substrates MBP and histone in the presence of H-MursiF or H-WKD, despite attempts to standardize reaction con- ditions and a longer exposure of the autoradiogram. Purified M. tuberculosis UsfX also does not display autophosphorylation or phosphotransfer to VSD. Also, MursiF WKD and UsfX failed to phosphorylate certain other anti-anti-SigF proteins, G-RsfB and G-Rv2638 (Table 1) (data not shown). In the auto- radiogram with both GST-tagged UsfX and WKD preparations, an autophosphorylating kinase signal was seen at a size lower than that of UsfX and WKD (Fig. S2). This autophosphorylation signal and a low- intensity signal for phosphotransfer on MBP seen in Fig. S2 are not unique to the presence of UsfX or WKD, as they are observed in the absence of these proteins but with GST protein and other recombinant nonkinase proteins, similarly purified from E. coli (Fig. S2). Most notably, autophosphorylation as well as phosphotransfer signals diminished after stringent and extensive washing of resin-bound G-UsfX and G-WKD with high salt (1 m NaCl) containing buffers before protein elution (Fig. S2). The activity was there- fore attributed to copurifying contaminating kinase(s) from E. coli. Two independent groups have already reported that M. tuberculosis UsfX, unexpectedly, is impaired in its ability to phosphorylate its natural sub- strates, anti-anti-sigma factors such as RsfA [10] and Rv0516c [30]. MursiF WKD and UsfX were tested for their ability to bind ATP by using a fluorescent ATP analog, 2,4,6- trinitrophenyl ATP (TNP-ATP), which shows an increase in fluorescence emission intensity accompanied by a blue shift upon protein binding [36]. The fluores- cence emission spectrum of TNP-ATP in the presence of MursiF WKD and UsfX showed a small change in 0 1 2 3 No inhibitor Sod.orthovanadate Cyclosporine Calyculin A Sod. flouride Okadaic acid A 405 Fig. 7. Effect of various phosphatase inhibitors on MursiF phospha- tase activity. Purified H-MursiF was preincubated with inhibitor (sodium orthovanadate, 200 l M; sodium fluoride, 50 mM; okadaic acid, 100 l M; cyclosporine, 5 mM; calyculin A, 1 lM) for 20 min at 25 °C in assay buffer prior to addition of pNPP. A 405 nm for each of the reactions at 100 min is plotted. Each value is the average of three individual reactions and is given as mean ± SD. P. Sachdeva et al. Functional characterization of Rv1364c FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS 6303 intensity as well as in wavelength of emission, whereas the positive control, M. tuberculosis PknB, showed a remarkable two-fold enhancement of the emission intensity and a blue shift (from 556 to 545 nm) (Fig. 8). M. tuberculosis MursiF WKD and UsfX therefore show very weak ATP-binding ability. Given this relatively low affinity of MursiF WKD and UsfX, low intracellular ATP concentrations in normal physi- ological conditions, and even lower ATP levels in the hypoxic and nonreplicating state in mycobacteria [37], their association with ATP in the cellular milieu for kinase function is questionable. To rule out loss of activity of purified MursiF WKD and UsfX during purification or handling, and to check for the presence of other features characteristic of anti- sigma factors, we carried out interaction studies with MursiF VSD and SigF using sandwich ELISA and an Ni 2+ -nitrilotriacetic acid resin pull-down assay, respec- tively (Fig. 9A,B). MursiF WKD was found to interact with both MursiF VSD (Fig. 9A) and SigF (Fig. 9B), in agreement with the results of Parida et al. [13]. The interaction between MursiF WKD and SigF is specific, as no interaction could be observed with the principal M. tuberculosis sigma factor, sigma factor A (SigA) (Fig. S3). Both MursiF WKD and UsfX show inter- molecular interactions with themselves as well as with each other (Fig. 9A). Similarly, UsfX interacts with 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 A B Blank H-VSD G-WKD GST G-WKD G-UsfX G-VSD GST G-UsfX G-WKD GST H-VSD Blank H-VSD H-VSD H-WKD H-VSD H-UsfX H-UsfX H-UsfX H-UsfX H-UsfX A 490 Added Coated H-SF bound – – – + + + resin G-UsfX G-WKD GST G-UsfX G-WKD GST 40% Input Output H-SF Fig. 9. Interactions of MursiF domains with each other and with UsfX (A) and SigF (B). (A) In a sandwich ELISA-based assay, the GST- tagged proteins (Table 1) were coated (Coated) on ELISA plates, and incubated with different concentrations of His-tagged interacting pro- teins (Added). The bound proteins were probed with antibody against penta-His and developed using ortho-phenylenediamine. GST served as negative control in the reactions. Each value is the average of three individual reactions and is given as mean ± SD. (B) Ni 2+ –Nitrilotriace- tic acid resin-bound H-SF was incubated with purified GST, G-WKD and G-UsfX in separate reactions, washed extensively, and analyzed by SDS ⁄ PAGE. Input lanes represent 40% of actual input protein added to H-SF-bound resin, and output lanes represent resin-bound proteins obtained in the interaction assay. 0 500 000 1 000 000 1 500 000 2 000 000 2 500 000 450 500 550 600 650 700 Emission wavelength (nm) Fluorescence intensity 1 2 3 4 Fig. 8. Fluorescence spectra of TNP-ATP in the presence and absence of MursiF. Details of the experiment are given in Experi- mental procedures. Curve 1: spectrum of TNP-ATP (8 l M) in the presence of buffer alone. Curve 2: TNP-ATP (8 l M) in the presence of H-WKD (1 l M). Curve 3: TNP-ATP (8 lM) in the presence of H-UsfX (1 l M). Curve 4: TNP-ATP (8 lM) in the presence of PknB (1 l M). Functional characterization of Rv1364c P. Sachdeva et al. 6304 FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS [...]... of phosphorylation, some other RsbW-like serine kinase may fill this missing link in the phosphorylation-dependent regulation of SigF by MursiF and UsfX in the Mycobacterium genus Further work to identify protein kinases that can compensate for this loss of activity of mycobacterial anti-sigma factors is in progress Experimental procedures In silico analysis The completely sequenced genomes of the Mycobacterium. .. scanned in the 470–650 nm range TNP-ATP binding was determined as the increase in fluorescence at 555 nm in the presence of protein All spectra were corrected for buffer fluorescence and for dilution (never exceeding 5% of the original volume) Sandwich ELISA The GST-tagged proteins were coated on ELISA plate (Maxisorb, Nunc, Naperville, IL, USA) in 50 mm carbonate buffer overnight at 4 °C The excess of protein. .. and anti-anti-sigma factor domains but has lost its kinase activity The absence of critical residues and deletions in MursiF and UsfX proteins across different species of Mycobacterium could indicate that functional dissection of kinase and anti-sigma factor activities is a phenomenon that is conserved across members of the Mycobacterium genus As MursiF has phosphatase activity and, as per our results,... carbonate buffer overnight at 4 °C The excess of protein was washed out with NaCl ⁄ Pi containing 0.05% Tween-20 (PBST), and wells were saturated with NaCl ⁄ Pi containing 1% BSA (PBSB) for 2 h at room temperature After six washes with PBST, the coated proteins were incubated with different concentrations of His-tagged interacting proteins in the presence of binding buffer (50 mm Tris, pH 7.4, 200 mm NaCl,... staining As a control, G-WKD and G-UsfX were incubated with the resin lacking H-SF under the same conditions to check for FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology Journal compilation ª 2008 FEBS P Sachdeva et al Functional characterization of Rv1364c nonspecific binding of the protein to the Ni2+–nitrilotriacetic acid 7 GST pull-down assay for interaction of. .. alignment J Mol Biol 302, 205–217 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 Chopra P, Singh B, Singh R, Vohra R, Koul A, Meena LS, Koduri H, Ghildiyal M, Deol P, Das TK et al (2003) Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB Biochem Biophys Res Commun 311, 112–120... WKD, rather than the antagonist domain, VSD M tuberculosis MursiF WKD and UsfX therefore show most of the attributes of functional anti-sigma factor proteins except for kinase activity In summary, we have demonstrated that the putative regulator of M tuberculosis SigF, MursiF, has retained phosphatase activity and its specific interaction with SigF as well as the interaction between its putative anti-sigma... bound to 100 lL of GST resin (1 h at 4 °C with rotation) and incubated with 7 lg each of H-SF and H-SA in binding buffer (50 mm Tris, pH 7.4, 200 mm NaCl, 0.1% NP-40, 10% glycerol) (100 lL) at 4 °C for 30 min with constant agitation After washing with 1 mL of wash buffer (50 mm Tris, pH 7.4, 300 mm NaCl, 0.1% NP-40, 10% glycerol) five times, the resin containing bound proteins was resuspended in SDS sample... LC, McAdam RA & Duncan K (2002) Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling Mol Microbiol 43, 717–731 Parida BK, Douglas T, Nino C & Dhandayuthapani S (2005) Interactions of anti-sigma factor antagonists of Mycobacterium tuberculosis in the yeast two-hybrid system Tuberculosis (Edinb) 85, 347–355 Cole ST, Eiglmeier K, Parkhill... described previously [42] The kinase reaction contained 2 lg of each enzyme in the kinase buffer (50 mm Tris ⁄ HCl, pH 7.6, 50 mm KCl, and 10 mm MgCl2, with and without 10 mm MnCl2, 1 mm dithiothreitol, and 0.1 mm EDTA) with 10 lg of each substrate in the presence of 5 lCi of [32P]ATP[cP] (BRIT, Hyderabad, India) and 20 lm unlabeled ATP in a total volume of 25 lL, and incubated for 30 min at 25 °C The reactions . Loss of kinase activity in Mycobacterium tuberculosis multidomain protein Rv1364c Preeti Sachdeva 1,2 , Azeet Narayan 1 , Richa Misra 1 , Vani Brahmachari 2 and Yogendra Singh 1 1 Institute of. stringent specificity of this group of phosphatases. MursiF WKD characterization To examine whether MursiF retains kinase activity in spite of the loss of critical residues involved in ATP binding,. and anti-anti-sigma factor, the loss of kinase activity of Rv1364c and UsfX appears to form a missing link in the phosphorylation-dependent interac- tion involved in SigF regulation in Mycobacterium. Abbreviations GST,