Correspondence: Jeong-Il Oh, Department of Microbiology, Pusan National University, 609-735 Busan, Korea. Tel.: +82-51-510-2593; fax: +82-51-514-1778; e-mail: firstname.lastname@example.org
The DevSR two-component system in Mycobacterium smegmatis consists of the DevS histidine kinase and the DevR response regulator. It is a regulatory system that is involved in the adaptation of mycobacteria to hypoxic and NO stresses. Using the yeast two-hybrid assay and pull-down assay, it was demonstrated that the phosphoaccepting Asp (Asp54) of DevR is important for protein–protein interactions between DevR and DevS. The negative charge of Asp54 of DevR was shown to play an important role in protein–protein interactions between DevR and DevS. When the Lys104 residue, which is involved in transmission of conformational changes induced by phosphorylation of the response regulator, was replaced with Ala, the mutant form of DevR was not phosphorylated by DevS and functionally inactive in vivo. However, the K104A mutation in DevR only slightly affected protein–protein interactions between DevR and DevS.
The two-component system (TCS) consisting of a homodimeric histidine kinase (HK) and its cognate response regulator (RR) is a simple signal transduction pathway that is used to elicit an appropriate adaptive response to environmental changes (Stock et al., 2000). The typical class I HK is usually a membrane-associated protein composed of the N-terminal sensory domain and the C-terminal core kinase domain that can be further divided into the dimerization and histidine phosphotransfer (DHp) and the CA (catalytic and ATP-binding) domains (Dutta et al., 1999). The DHp domain consists of ~70 amino acid residues that form a secondary structure of α1-helix-loop-α2-helix (Tomomori et al., 1999; Marina et al., 2005; Casino et al., 2009) and serves as an interacting domain of HK with RR (Tomomori et al., 1999; Seok et al., 2006; Skerker et al., 2008; Casino et al., 2009). The domain structure of the typical RR is divided into the N-terminal regulatory domain and the C-terminal effector domain that are connected by the short linker (Stock et al., 2000). The regulatory domain contains a conserved Asp residue that receives the phosphoryl group from an autophosphorylated HK. Six residues (three acidic amino acids, one Thr/Ser, one Tyr/Phe, and one Lys), which are functionally important for phosphorylation of the RR and the activation of the effector domain through phosphorylation-induced conformational changes, are conserved in the regulatory domain (Fig. 1a) (Stock et al., 1989b; Bourret et al., 1990; Lukat et al., 1991). The structure of the typical RR regulatory domain has the canonical (βα)5 fold (Stock et al., 1989a; Volz & Matsumura, 1991). The two consecutive acidic amino acids directly or indirectly coordinating a Mg2+ ion and the phosphorylatable Asp are located in the β1-α1 and β3-α3 loops, respectively (Lukat et al., 1991; Stock et al., 1993). The conserved Lys residue is located in the β5-α5 loop and forms a salt bridge with phosphoryl oxygen of phosphoaspartate in the phosphorylated RR. It was demonstrated that the α1-helix and the β5-α5 loop of the RR regulatory domain directly interact with the α1-helix of the HK DHp domain (Zapf et al., 2000; Casino et al., 2009).
The DevSR TCS of M. smegmatis is composed of the DevS HK (MSMEG_5241) and its cognate DevR RR (MSMEG_5244) (Mayuri Bagchi et al., 2002). The DevSR TCS and its homolog in Mycobacterium tuberculosis (MTB), DosSR (also called DevSR) TCS, were shown to play an important role in the adaptation of mycobacteria to hypoxic and NO conditions where physiology of mycobacteria is altered to shift into the nonreplicating dormant state (Park et al., 2003; Roberts et al., 2004). The DosT HK, which is a functional paralog of DosS, is also responsible for phosphorylation and dephosphorylation of DosR in MTB (Roberts et al., 2004; Saini et al., 2004a). There is a second set of the genes for DevS (MSMEG_3941) and DevR (MSMEG_3944) in M. smegmatis. However, they appear not to be functional in terms of hypoxic induction of the genes that are under control of DevR, as the hspX gene, which is regulated by the DevSR TCS, is induced in neither MSMEG_5241 nor MSMEG_5244 mutants grown under hypoxic conditions (Kim et al., 2010; see Fig. 3b).
The three-dimensional structure of unphosphorylated DosR of MTB revealed that DosR assumes an atypical folding structure that is different from those of other typical RRs (Fig. 1b). DosR has a (βα)4 folding structure in place of the canonical (βα)5 fold and the relatively long linker region consisting of two α-helices (α5 and α6) (Wisedchaisri et al., 2008). The conserved Lys residue (Lys104) of DosR, whose counterpart is positioned in the β5-α5 loop in other RRs, is located at the beginning of the α5-helix in the linker region. As a result, the Lys104 residue is far from the active site in the DosR structure. Due to the high identity (85% identity) between DosR and DevR and functional complementarity of DosS and DosT in a DevS mutant of M. smegmatis (Kim et al., 2010), DevR of M. smegmatis is predicted to assume the structure similar to DosR of MTB.
We previously reported that the yeast two-hybrid (Y2H) assay is a useful tool for the detection of the specific protein interaction between a HK and its cognate RR (Seok et al., 2006; Lee et al., 2012). Using Y2H assay, we herein report the involvement of the phosphorylatable Asp (Asp54) of DevR in protein–protein interactions between DevR (MSMEG_5244) and DevS (MSMEG_5241) and importance of the conserved Lys104 in DevR function.
Materials and methods
Strains, plasmids, and general molecular techniques
Escherichia coli, Saccharomyces cerevisiae, and M. smegmatis strains as well as plasmids used in this study are listed in Table S1 in Supporting Information. Growth conditions of bacterial and yeast strains, transformation of yeast and M. smegmatis strains, construction of a devR (MSMEG_5244) deletion mutant of M. smegmatis, site-directed mutagenesis, immunoblotting, protein determination, and reverse transcription (RT) PCR are described in Data S1.
Analysis of in vivo protein–protein interactions
Saccharomyces cerevisiae AH109 strain co-transformed with both pGADT7 and pGBKT7 derivatives was grown in synthetic defined dropout (SD) medium (Q-Biogene, Canada) lacking leucine and tryptophan (SD/-Leu/-Trp). The overnight cultures were diluted to an optical density at 600 nm (OD600 nm) of 0.6 and spotted onto both solid SD/-Leu/-Trp medium and SD/-Leu/-Trp/-His medium containing appropriate concentrations of 3AT (3-amino-1,2,4-trizole: Sigma, MD). These plates were incubated for 3 to 5 days. Determination of β-galactosidase activity was performed using o-NPG as a substrate as described elsewhere (Schneider et al., 1996).
Truncated DevS: The truncated DevS (MSMEG_5241) lacking the N-terminal sensory domain (DevSc: amino acids from 343 to 571) was overexpressed in an E. coli BL21 (DE3) strain carrying pT7STHIS and purified as described previously (Lee et al., 2008).
GST-DevR fusion proteins: GST-DevR (MSMEG_5244) fusion proteins were overexpressed in E. coli BL21 (DE3) strain harboring the corresponding pT7GST derivatives. The strain was grown aerobically at 37 °C in LB medium containing 100 μg mL−1 ampicillin to an OD600 nm of 0.4 to 0.6. The devR gene was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM, and the cells were further grown for 4 h at 30 °C. Harvested cells from 2 L culture were resuspended in 10 mL PBS buffer [140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 (pH 7.3)] and disrupted by two passages through a French pressure cell. Following DNase Ι treatment (10 units mL−1) in the presence of 10 mM MgCl2 for 30 min on ice, cell-free crude extracts were obtained by centrifugation twice at 20 000 g for 15 min. One mL of the 50% (v/v) slurry of Glutathione Sepharose 4B resin (Amersham Biosciences, NJ) was added to the crude extracts and mixed gently by shaking for 1.5 to 2 h on ice. The protein-resin mixture was packed into a column, and the resin was washed with 40 bed volumes of PBS buffer, and then, GST-DevR was eluted with 10 bed volumes of elution buffer [10 mM reduced glutathione in 50 mM Tris-HCl (pH 8.0)]. The fractions from the elution step were collected and dialyzed against 2 L of 20 mM Tris-HCl (pH 8.0) overnight at 4 °C. The dialyzed protein was concentrated using a centrifugal filter device (Millipore, MA).
In vitro kinase assay
Autophosphorylation of DevSc was performed at 30 °C in an assay buffer [20 mM Tris-HCl (pH 8.0), 50 mM KCl, 5 mM MgCl2] in the presence of a mixture of [γ-32P]ATP and unlabeled ATP to a final concentration of 200 μM (500 Ci mol−1) for 1 h. After autophosphorylation reaction, 250 pmol of DevSc was added to the wild-type and mutant forms of GST-DevR (100 pmol) for phosphotransfer reactions (the total reaction volume was 20 μL). The reactions were performed at room temperature for 1.5 min and stopped by the addition of 5 μL of 5× loading buffer [50 mM Tris-HCl (pH 6.8), 4% (w/v) sodium dodecyl sulfate (SDS), 20% (w/v) glycerol, 20 mM dithiothreitol, 1% (v/v) β-mercaptoethanol, 0.1% (w/v) bromophenol blue, and 100 mM EDTA]. The phosphorylation level of the proteins was determined by SDS-PAGE and subsequent autoradiography.
The assay was performed using Ni-sepharose high-performance resin (Amersham Biosciences) to detect physical interactions between His6-tagged DevS and GST-DevR. Five nmol of DevSc was mixed with 5 nmol of each GST-DevR (wild-type, D54A, or K104A DevR) and incubated for 10 min at room temperature (the total mixture volume was 200 μL). 50 μL of the 50% (v/v) resin was added to the DevSc-DevR mixtures, and the resin-protein mixtures were incubated for 10 min at room temperature. The mixtures were centrifuged at 160 g. at a benchtop centrifuge for 1.5 min, and the supernatants were removed by pipetting. The resin pellets were resuspended in 400 μL of wash buffer [20 mM Tris-HCl (pH 8.0) containing 20 mM β-mercaptoethanol] by gently tapping the tubes, and then, the tubes were centrifuged at 160 g for 1.5 min. The wash step was repeated twice. For elution of proteins from the resin, the washed resin pellets were resuspended in 100 μL of elution buffer [20 mM Tris-HCl (pH 8.0) containing 500 mM imidazole], and the resuspended resin pellets were incubated for 10 min at room temperature. The resin pellets were centrifuged at 160 g for 2 min. Supernatants were taken and concentrated under vacuum. The concentrated samples were analyzed by SDS-PAGE.
The effect of D54A and K104A mutations in DevR on protein–protein interactions between DevR and DevS
As shown in Fig. 2a, we previously demonstrated by means of Y2H assay using the PrrA RR and its cognate PrrB HK of Rhodobacter sphaeroides that mutations of the phosphoaccepting Asp63 and Lys113 residues among the amino acid residues (Asp19, Asp20, Asp63, Tyr91, and Lys113) constituting the active site of PrrA abolished protein–protein interactions between PrrA and PrrB (Seok et al., 2006). Western blotting analysis showed that the approximately same amounts of the GAL4AD-PrrA, GAL4AD-D63A PrrA, and GAL4AD-K113A PrrA fusion proteins were detected in the yeast strains, indicating that abolishment of protein interactions for the mutant forms of PrrA did not result from the decreased expression or degradation of the corresponding GAL4AD-PrrA fusion proteins.
To confirm this finding, the effect of D54A and K104A mutations in DevR on protein–protein interactions between DevR and DevS was investigated (Fig. 2b). Consistent with the result of the PrrBA pair, no protein–protein interactions were detected in Y2H assay when the phosphoaccepting Asp residue, Asp54, was replaced with Ala. However, Y2H assay showed that the K104A mutation in DevR did not abolish protein–protein interactions between DevR and DevS unlike the PrrBA system, but slightly reduced the extent of the protein interaction. Pull-down assay using the purified C-terminally GST-fused DevR and C-terminally His6-tagged DevS revealed that the amount of D54A DevR pulled down by DevS was substantially reduced compared with the wild-type DevR, which is in good agreement with the result obtained from Y2H assay (Fig. 2c). The pull-down assay also revealed that the abolishment of the protein interaction of D54A DevR with DevS was not a consequence of the reduced GAL4AD-DevR fusion protein expressed in the yeast strain (see Fig. 4a). Furthermore, this assay also confirmed that protein–protein interactions between K104A DevR and DevS were only slightly affected.
The K104A mutant form of DevR is not phosphorylated by DevS and functionally inactive in vivo
The ability of K104A DevR to receive a phosphoryl group from autophosphorylated DevS was investigated. The wild-type DevR and D54A DevR were included in the phosphotransfer reactions as the positive and negative controls, respectively. As in the pull-down assay described above, the GST-fused DevRs and His6-tagged DevS were used in the experiment. When the wild-type DevR was added to the autophosphorylated DevS and the phosphotransfer reaction was performed for 1.5 min, the level of the phosphorylated DevS was significantly decreased and the band of the phosphorylated DevR appeared (Fig. 3a), indicating transfer of a phosphoryl group from DevS to DevR. In contrast, the addition of the K104A and D54A mutant forms of DevR to the autophosphorylated DevSc did not change the level of the phosphorylated DevS that was same as that of the phosphotransfer reaction without DevR. This result indicates that Lys104 plays an important role in phosphorylation of DevR despite its atypical location in the regulatory domain.
We next examined whether or not K104A DevR is functional in M. smegmatis. The DevSR TCS is responsible for the hypoxic induction of many genes belonging to the DevR regulon. The hspX gene encoding α-crystallin is one of the DevR regulon genes that is greatly induced under hypoxic conditions (Sherman et al., 2001). It was determined by complementation tests whether K104A DevR is functional in M. smegmatis. A devR (MSMEG_5244)-deletion mutant strain of M. smegmatis (ΔdevR) was complemented by the introduction of the pNBV1 shuttle vector carrying the wild-type devR gene or the mutant devR gene, and complementation was judged by the hypoxic induction of hspX (Fig. 3b). As expected, the ΔdevR strain of M. smegmatis containing the empty vector did not show the induction of hspX under hypoxic conditions (Control), whereas the mutant strain harboring the wild-type devR gene in trans exhibited the strong induction of hspX under hypoxic conditions. Hypoxic induction of hspX was not observed in the ΔdevR strains with either D54A DevR or K104A DevR.
The negative charge of Asp-54 is important for protein–protein interactions between DevR and DevS
Y2H assays using PrrA and DevR demonstrated that the phosphoaccepting Asp residue in the regulatory domain is essential not only for phosphorylation of RR but also for protein–protein interactions between RR and HK (Seok et al., 2006). To determine whether the negative charge of the Asp residue is important for the protein interaction, we replaced the Asp54 residue with either Glu or Asn and examined protein–protein interactions between DevR and DevS by means of Y2H assay (Fig. 4). Protein–protein interactions were not detected between D54N DevR and DevS, whereas D54E DevR resulted in more robust protein interactions with DevS than the wild-type DevR. To determine whether the expression and/or stability of the GAL4AD-DevR fusion proteins in yeast cells were affected by these mutations, we performed Western blotting analysis with a monoclonal antibody against GAL4AD. As shown in Fig. 4a, it was demonstrated that the lack of the protein interaction between D54N DevR and DevS in Y2H assay did not result from instability or degradation of the D54N DevR-GAL4AD fusion protein and that the strengthened protein interaction between D54E DevR and DevS was not caused by an increase in the D54E DevR-GAL4AD fusion protein.
This study was carried out to assess the role of the phosphoaccepting Asp residue (Asp54) of DevR in protein–protein interactions between DevR and its cognate DevS HK. Previously, we demonstrated by means of Y2H assay that replacement of the phosphorylatable Asp with Ala in the PrrA RR of R. sphaeroides abolished the protein interaction with the PrrB HK (Seok et al., 2006). The strength of protein–protein interactions between PrrA and PrrB was so weak that we did not prove the importance of the Asp residue in protein–protein interactions using experimental approaches other than Y2H assay. As DevR was shown to interact with DevS more strongly in Y2H assay than the PrrA-PrrB pair (Seok et al., 2006), we could perform the pull-down assay using the purified DevR-DevS pair in addition to Y2H assay to confirm our previous finding. As with the two-component PrrBA, replacement of Asp54 with Ala in DevR completely abolished protein interactions of DevR with DevS in Y2H assay, which was further confirmed in the pull-down assay. These findings strongly indicate that the Asp residue of an RR, which receives a phosphoryl group from its cognate autophosphorylated HK, is essential not only for phosphorylation of the RR but also for protein–protein interactions between RR and HK. The negative charge of the Asp residue appears to be important for protein interactions of the RR with its partner HK, as judged by the finding that D54N DevR did not interact with DevS. It was reported that the D54N mutant form of MTB DosR was not phosphorylated by DosS, implying that the D54N DevR of M. smegmatis is defective in both protein interaction with DevS and phosphorylation by DevS (Saini et al., 2004b). To the best of our knowledge, this is the first report demonstrating involvement of the phosphoaccepting Asp residues of RRs in protein interactions with their cognate HKs. Replacement of the phosphoaccepting Asp residue with Glu revealed rather stronger protein interactions between DevR and DevS, which implies that phosphorylation of DevR might lead to stronger protein–protein interactions between DevR and DevS, as the length of the side chain and the presence of the negative charge of the phosphorylated Asp are similar to Glu. The effect of RR phosphorylation on its protein interaction with HK has been investigated for the EnvZ-OmpR TCS of E. coli by two research groups (Mattison & Kenney, 2002; Yoshida et al., 2002). Both groups yielded the contradicting results. One group showed the reduced affinity of the phosphorylated OmpR for EnvZ compared to unphosphorylated OmpR. The other group revealed that OmpR affinity for EnvZ was similar to that of the phosphorylated OmpR. It was demonstrated that the D54E mutant form of MTB DosR was defective in phosphotransfer from DosS to DosR (Roberts et al., 2004), indicating that the conservative substitution of Asp54 by Glu adversely affects not the protein interaction of DevR with DevS, but the phosphotransfer reaction from the autophosphorylated DevS to DevR.
We also performed in vivo and in vitro approaches to reveal the functional importance of Lys104 in protein–protein interactions between DevR and DevS. In contrast to PrrA, the K104A mutation in DevR did not significantly alter protein–protein interactions between DevR and DevS. This difference can be explained by the unusual folding structure of the DevR regulatory domain (Wisedchaisri et al., 2008). As DosR of MTB and DevR of M. smegmatis share 85% identity at the level of their primary structures, DevR is likely to have the similar three-dimensional structure as DosR. Although Lys104 appears to be positioned far away from the active site for phosphorylation due to the different folding structure of the DevR regulatory domain (see Fig. 1b), our results demonstrated that Lys104 is still important for phosphorylation of DevR and its functionality. The functional importance of Lys104 with regard to phosphorylation of DosR was also reported for MTB DosR (Saini et al., 2004b).
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0015918).