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1 Department of Microbiology, Pusan National University, 609-735 Busan, South Korea
2 Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center Medical School, 6431 Fannin, Houston, TX 77030, USA
Correspondence
Jeong-Il Oh
joh{at}pusan.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-helix 1 and its flanking turn region within the dimerization domain (DD) of the PrrB histidine kinase appear to play an important role in conferring the recognition specificity for the PrrA response regulator on the DD. The catalytic ATP-binding domain of the histidine kinase, which functions as the catalytic unit for the phosphotransfer reaction from ATP to the conserved histidine residue in the DD, also appears to contribute to the enhancement of the recognition specificity conferred by the DD. It was also revealed that replacement of Asp-63 and Lys-113 of the PrrA response regulator by alanine abolished protein–protein interactions between PrrA and its cognate histidine kinase PrrB, whereas mutations of Asp-19, Asp-20 and Thr-87 to alanine did not affect protein–protein interactions, indicating that among the active site residues of PrrA, Asp-63 and Lys-113 are important not only in the function of PrrA but also for protein–protein interactions between PrrA and PrrB.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; West & Stock, 2001). The typical two-component system consists of a histidine kinase and a cognate response regulator. The orthologous form of the histidine kinase comprises the N-terminal sensing domain responsible for signal sensing and the C-terminal transmitter domain that is involved in ATP-dependent phosphorylation of the cognate response regulator. The transmitter domain is further divided into the dimerization domain (DD) and the catalytic ATP-binding (CA) domain (Dutta et al., 1999). The DD consists of 
70 amino acid residues that form a secondary structure of -helix–turn–-helix (Tomomori et al., 1999). Histidine kinases exist as homodimers, in which the DDs of two identical subunits form a four-helix bundle. In the middle of the first (N-terminal) -helix of the DD a histidine residue is conserved among all histidine kinases and the histidine residue serves as the autophosphorylation site. The CA domain exhibits a structural homology with ATPases such as Hsp90, DNA gyrase B and MutL, and contains conserved signatures of histidine kinases (N, G1, F and G2 boxes), which form the unique ATP-binding pocket (Dutta et al., 1999). This domain is responsible for the phosphotransfer reaction from ATP to the histidine residue conserved in the DD. The CA domain of one subunit is juxtaposed to the DD of the other subunit. Due to the highly flexible interdomain hinges between CA and DDs, the 3D structure of the intact transmitter domain of a histidine kinase is yet to be solved. However, the structures of each independent domain of the histidine kinase have been resolved (Tanaka et al., 1998; Tomomori et al., 1999).
The response regulator usually has a two-domain structure, with a conserved N-terminal regulatory domain and a variable C-terminal effector domain (West & Stock, 2001). The regulatory domain is also called the receiver domain and contains a conserved aspartate residue that receives the phosphoryl group from a phosphorylated histidine kinase. The phosphorylation of the regulatory domain brings about the conformational change, which leads to the activation of the effector domain that usually functions as the DNA-binding domain. Many histidine kinases are bifunctional enzymes that have both kinase and phosphatase activities acting on their cognate response regulators (Stock et al., 2000; Oh & Kaplan, 2001; Oh et al., 2004). Depending on the presence or absence of an environmental signal, the equilibrium of the kinase/phosphatase activities of a histidine kinase is regulated, and this in turn controls the cellular abundance of the phosphorylated response regulator that forms the phosphorelay couple with the histidine kinase.
The photosynthetic bacterium Rhodobacter sphaeroides can grow in various environments and has been shown to possess extensive metabolic capability (Kiley & Kaplan, 1988; Zeilstra-Ryalls et al., 1998; Oh & Kaplan, 2001). In order to adapt to various environmental conditions, R. sphaeroides possesses approximately 30 different pairs of two-component systems, excluding the CheA-CheY chemotaxis systems (www.rhodobacter.org). In organisms with multiple two-component systems, signal transduction through these two-component systems requires precise interactions between histidine kinases and their cognate response regulators to elicit the correct response in adaptation to a given environmental signal. Currently little is known about the mechanisms underlying the specific interaction between a histidine kinase and a cognate response regulator. Using the PrrBA and DevSR two-component systems that are involved in redox sensing in R. sphaeroides and Mycobacterium smegmatis (Eraso & Kaplan, 1994, 1995; Mayuri et al., 2002), respectively, we performed in vivo mapping of the interaction domains of the histidine kinases and their cognate response regulators by means of yeast two-hybrid assays, and revealed regions (domains) on the histidine kinases that confer recognition specificity for their cognate response regulators.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. E. coli strains were grown at 37 °C in Luria–Bertani (LB) medium as described elsewhere (Sambrook et al., 1989). Ampicillin (100 µg µl–1) and kanamycin (50 µg µl–1) were added when appropriate. S. cerevisiae strains were grown at 30 °C in YPD (Clontech) or synthetic defined dropout (SD) medium (Q-Biogene) lacking adenine, histidine, leucine and tryptophan (SD/–Ade/–His/–Leu/–Trp) in the presence of different concentrations of 3-amino-1,2,4-trizole (3AT; Sigma) as described in the manufacturer's manual (www.clontech.com/clontech/techinfo/manuals/PDF/PT3024-1.pdf).
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). S. cerevisiae strains were cotransformed with different pairs of two-hybrid plasmids according to the lithium acetate (LiAc)-mediated method as described elsewhere (Guthrie & Fink, 1991).
Construction of plasmids
i) pPLA and pBBC.
To construct pPLA, a 0.64 kb EcoRI–BamHI fragment containing prrA was amplified with the primers PrrA(EcoRI)+ (5'-ccttgaattccgaatgacagccatgtgtc-3') and PrrA(BamHI)– (5'-cttcggatcctcagcgcgggctgcgcttc-3') using the plasmid pUI1643 as the template and pfu DNA polymerase. The PCR product was restricted with EcoRI and BamHI and cloned into pGADT7 encoding the GAL4 activation domain (GAL4AD) digested with the same restriction enzymes, yielding pPLA. Most prrA derivatives were cloned into pGADT7 by the same procedure as described above to produce the fusion proteins (GAL4AD–PrrA derivatives). To construct pBBC, a 0.91 kb BamHI–PstI fragment containing a portion of prrB was amplified by PCR using the primers PrrBc(BamHI)+ (5'-tataggatccgcatgttcgaattcggc-3') and PrrB(PstI)– (5'-atatctgcagatcaggtctggatcagg-3'), digested with BamHI and PstI, and cloned into pGBKT7, giving pBBC. Most PrrB derivatives were fused to the C terminus of the GAL4 DNA-binding domain (GAL4BD) employing the vector pGBKT7.
ii) pPLR and pBSC.
A 0.74 kb EcoRI–BamHI fragment containing devR was amplified by PCR using the primers DevR(EcoRI)+ (5'-atgcgaattcaccccgcggctctgggtgcgc-3') and DevR(BamHI)– (5'-atgcggatccctagttgcgccggtccagttt-3') from M. smegmatis MC2 chromosomal DNA as the template. A 0.69 kb EcoRI–BamHI fragment containing a portion of devS was amplified with the primers DevSc(EcoRI)+ (5'-atgcgaattccctttcaccgcagaacagttg-3') and DevS(BamHI)– (5'-atgcggatcctcagtcggggagcggcgcggt-3') by the same procedure. The DNA fragments containing devR and a portion of devS were digested with EcoRI and BamHI and cloned into pGADT7 and pGBKT7 restricted with the same restriction enzyme, yielding pPLR and pBSC, respectively.
iii) pBBSDI and pBBSDII.
A recombinant PCR method was performed to generate PrrB–DevS chimeric proteins. Two rounds of PCR were carried out using pfu polymerase. The plasmids pUI1643 and pBSC were used as the templates for amplification of portions of prrB and devS, respectively. Two primary PCR reactions were performed with the primers PrrBc(BamHI)+ and BSI– (5'-ctgctgcacctcggccgaacgaagctcctcggcaagctccg-3') and with BSI+ (5'-cggagcttgccgaggagcttcgttcggccgaggtgcagcag-3') and DevS(BamHI)– (5'-atgcggatcctcagtcggggagcggcgcggt-3') to generate two DNA fragments each containing a 42 bp overlapping region. Two primary PCR products were used as the templates for the secondary PCR, which was performed using the primers PrrBc(BamHI)+ and DevSD(BamHI)– (5'-atatggatccatcgaagatggccgtgcg-3'). The secondary PCR product was restricted with EcoRI and BamHI and cloned into pGBKT7 digested with the same restriction enzymes to yield pBBSDI. The plasmid pBBSDII was constructed in the same way as pBBSDI, except for using the primers BSII+ (5'-cgaacgtgcgcgcgggatcgtcgagctcaccagcttgatcgt-3') and BSII– (5'-acgatcaagctggtgagctcgacgatcccgcgcgcacgttcg-3') in place of BSI+ and BSI–.
iv) pPR and pBZD.
To construct pPR, a 0.72 kb EcoRI–BamHI fragment containing ompR was amplified by PCR using the primers OmpR(EcoRI)+ (5'-atatgaattcatgcaagagaactacaag-3') and OmpR(BamHI)– (5'-atatggatcctcatgctttagagccgtc-3') from E. coli chromosomal DNA as the template. The PCR product was restricted with EcoRI and BamHI and cloned into pGADT7 digested with the same restriction enzymes, yielding pPR. A 0.21 kb EcoRI–BamHI fragment containing a portion of envZ was amplified with the primers EnvZDD (EcoRI)+ (5'-atatgaattcatggcggctggtgttaag-3') and EnvZDD (BamHI)– (5'-atatggatccctcctgcccggtgcgcag-3') by PCR, digested with EcoRI and BamHI, and cloned into pGBKT7, giving pBZD.
Site-directed mutagenesis.
To mutate codons corresponding to Asp-19, Asp-20, Asp-63, Thr-91 and Lys-113 of PrrA to alanine, mutagenesis was performed using the Quick Change Site-directed Mutagenesis kit (Stratagene) using pULAR as a template plasmid. Synthetic oligonucleotides 33 bases long containing an alanine codon (GCC, GCT, GCG) in place of the codon of the corresponding amino acid in the middle of their sequences were used to mutagenize the prrA gene. Following the verification of mutations by DNA sequencing, a 0.48 kb EcoRI–BamHI fragment containing the mutated sequence was cloned into pGADT7, giving pPLAR19, pPLAR20, pPLAR63, pPLAR91 and pPLAR113.
Analysis of in vivo protein–protein interactions.
The S. cerevisiae strains co-transformed with both pGADT7 and pGBKT7 derivatives were grown in SD/–Leu/–Trp medium. The cultures were diluted to OD600 0.4–0.45 and spotted onto both solid SD/–Leu/–Trp medium and SD/–Leu/–Trp/–His medium containing different concentrations of 3AT. These plates were incubated for 3–7 days. The crude cell extracts were prepared by three freeze/thaw cycles using liquid nitrogen. Determination of 
-galactosidase activity was performed using ONPG as substrate, as described elsewhere (Schneider et al., 1996).
Immunoblotting.
SDS-PAGE and Western blotting were performed as described elsewhere (Laemmli, 1970; Mouncey & Kaplan, 1998). The protein extracts were resolved by 12.5 % (w/v) SDS-PAGE and transferred to PVDF membranes. The mouse mAbs against GAL4AD (SantaCruz) and GAL4BD (BD Biosciences) were used at dilutions of 1 : 200 and 1 : 10 000, respectively. The alkaline phosphatase-conjugated anti-mouse IgG (Sigma) was used at a 1 : 10 000 dilution for the detection of the primary antibodies. Detection was carried out by staining the membrane with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Comolli et al., 2002; Oh et al., 2004). The DNA fragment which was cloned into the prey vector to produce a GAL4AD–PrrA fusion protein contained the 84 bp DNA sequence upstream of the prrA start codon in addition to the entire prrA gene. Since this additional DNA sequence does not contain a stop codon that lies in the same reading frame as the GAL4AD and prrA genes, the PrrA protein expressed from pPLAC (pGADT7 : : prrA) has an additional 28 amino acid residues at its N terminus. In order to assess the specificity of the interaction between the histidine kinase and its cognate response regulator using the yeast two-hybrid assay, another two-component system, the DevSR two-component system from M. smegmatis, was included. The genes for the DevS histidine kinase and DevR response regulator were cloned into the bait and prey vectors, respectively, in the same way as for the PrrBA two-component system (DevR of the GAL4AD–DevR fusion protein also has an additional 28 amino acid residues at its N terminus). When protein–protein interactions between GAL4AD and GAL4BD fusion proteins occur in the yeast strain AH109 carrying both bait and prey plasmids, the yeast strain is enabled to grow on histidine-deficient medium and synthesize -galactosidase due to the induction of the HIS3 and lacZ reporter genes on its chromosomal DNA. It was further possible to comparatively determine the strength of the protein–protein interactions by using various concentrations of 3AT, which serves as an inhibitor of the HIS3 product.
As shown in Fig. 1, the yeast strain (PrrA/PrrBc) expressing both the GAL4AD–PrrA and the GAL4BD–PrrBc fusion proteins grew on histidine-deficient medium (SD medium lacking histidine), indicating that PrrA interacts with PrrBc. Likewise, the yeast strain with the DevR/DevSc pair grew on SD medium without histidine, and its growth in the presence of 5 mM 3AT was more robust than that of the yeast strain (PrrA/PrrBc) under the same conditions. This result indicated that the protein–protein interactions between DevR and DevSc are stronger than those between PrrA and PrrBc. The yeast strains (PrrA/DevSc and DevR/PrrBc) which expressed heterologous pairs of fusion proteins did not grow on SD medium lacking histidine. Taken together, the data presented in Fig. 1 demonstrate that the yeast two-hybrid system used in this study is applicable for the study of the specific protein–protein interactions between a histidine kinase and its cognate response regulator.
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). The regulatory domain of PrrA contains an aspartate residue (Asp-63) that receives the phosphoryl group from autophosphorylated PrrB (Comolli et al., 2002), while the effector domain functions as the DNA-binding domain (Laguri et al., 2003).
As shown in Fig. 2(A), the yeast strain (PrrAr/PrrBc) expressing both GAL4AD–PrrAr (regulatory domain of PrrA) and GAL4BD–PrrBc fusion proteins grew on SD medium lacking histidine. The growth of the yeast strain (PrrAr/PrrBc) on histidine-deficient medium supplemented with 3AT was more robust than that of the yeast strain (PrrA/PrrBc) under the same growth conditions. In keeping with this observation, the -galactosidase assay revealed that a 1.9-fold higher activity of -galactosidase was detected in cell lysates of the yeast strain (PrrAr/PrrBc) compared to the yeast strain (PrrA/PrrBc). The PrrA derivative (PrrAe), in which the regulatory domain was removed from PrrA, did not interact with PrrBc in the yeast strain (PrrAe/PrrBc). The yeast strain (P/PrrBc) harbouring the empty prey vector (P) as a negative control did not grow on histidine-deficient medium. Taken together, these results indicate that the regulatory domain of PrrA is the region in which protein–protein interactions between PrrA and PrrB occur, and that the PrrA derivative (PrrAr) consisting of the regulatory domain alone appears to interact with PrrBc more strongly than does intact PrrA, suggesting a negative effect of the effector domain on this interaction.
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To determine whether the expression and/or stability of the GAL4AD–PrrA fusion proteins in yeast cells were affected by these deletions, we performed Western blotting analysis with a mAb against GAL4AD. As shown in Fig. 2(B)
, all GAL4AD fusion proteins were detected in the Western blotting assay, ruling out the possibility that the inability of some PrrA derivatives to interact with PrrBc might have resulted from instability or degradation of their fusion proteins. From the results it can be inferred that the intact form or conformation of the PrrA regulatory domain is required for the correct protein–protein interactions between PrrA and PrrB, and that it is necessary to insert a linker of the proper length between GAL4AD and PrrA when the GAL4AD–PrrA fusion proteins for the yeast two-hybrid assay are constructed.
D63A and K113A substitutions in the regulatory domain of PrrA abolish protein–protein interactions between PrrA and PrrB
The regulatory domains of response regulators have a (/)5 topology with five parallel -strands surrounded by five -helices (Volz & Matsumura, 1991; Stock et al., 1993). The active site located in the regulatory domain of the response regulator is believed to be composed of three acidic residues, a lysine residue and a hydroxyl amino acid (threonine or serine), which are all conserved in all known response regulators (Fig. 3A). The corresponding conserved active site residues in PrrA are Asp-19, Asp-20, Asp-63, Thr-91 and Lys-113. Asp-63 has been demonstrated to be the site of phosphorylation (Comolli et al., 2002). Asp-19 and Asp-20 are predicted to be involved in coordination of a Mg2+ ion that is essential for phosphorylation and dephosphorylation of the response regulator (Stock et al., 1993). It is assumed that Thr-91 and Lys-113 are involved in the phosphorylation-induced conformational change of the response regulator (Lukat et al., 1991; Appleby & Bourret, 1998).
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, the positive-control yeast strain (PrrAr/PrrBc) grew on histidine-deficient medium, while the yeast strain (P/PrrBc) carrying the empty prey vector did not grow on histidine-deficient medium. The mutant forms (PrrArD19A, PrrArD20A and PrrArT91A) of the PrrA regulatory domain in which Asp-19, Asp-20 and Thr-91, respectively, were replaced with alanine, were shown to interact with PrrBc to the same extent as the wild-type form of the PrrA regulatory domain (PrrAr), indicating that D19A, D20A and T91A mutations of PrrAr do not affect the protein–protein interactions between PrrAr and PrrBc to any significant degree. In contrast, replacement of Asp-63 or Lys-113 with alanine abolished protein–protein interactions between PrrAr and PrrBc, as judged by the inability of the corresponding yeast strains (PrrArD63A/PrrBc, PrrArK113A/PrrBc) to grow on histidine-deficient medium.
To determine whether the inability of the yeast strains expressing the mutant forms (D63A and K113A) of PrrAr to grow on histidine-deficient medium was the result of instability of the corresponding GAL4AD–PrrAr fusion proteins in the yeast cell, we performed Western blotting analysis with a mAb against GAL4AD. As shown in Fig. 3(C), all GAL4AD fusion proteins with molecular masses of 43 kDa were detected by Western blotting analysis, indicating that inability of the yeast strains (PrrArD63A/PrrBc, PrrArK113A/PrrBc) to grow on histidine-deficient medium was not the result of instability or degradation of the fusion proteins in the yeast cell.
The DD of PrrB is the region that interacts with PrrA and confers recognition specificity for interactions between PrrB and PrrA
The transmitter domain (PrrBc) of the PrrB histidine kinase, which catalyses the phosphotransfer reaction from ATP to the PrrA response regulator, is composed of two distinct domains, the DD and the CA domain (Bird et al., 1999; Comolli et al., 2002; Oh et al., 2004).
To examine the contributions of these individual domains to the PrrB interaction between the PrrB transmitter domain and PrrA, protein–protein interactions between the different truncated PrrBc derivatives and PrrA were analysed in the yeast two-hybrid assay. As shown in Fig. 4(A), the yeast strain (PrrA/PrrBc) expressing both GAL4AD–PrrA and GAL4BD–PrrBc fusion proteins grew on histidine-deficient medium, whereas the negative-control strain (PrrA/B) harbouring the empty bait vector (B) did not grow on the same growth medium. The PrrBc derivatives (PrrBc320, PrrBc270 and PrrBc267) in which 142, 192 and 195 amino acids were removed from the C terminus of PrrBc, respectively, retained the intact DD, but a part or the whole of the CA domain was deleted in these PrrBc derivatives. The PrrBc derivatives containing the intact DD interacted with PrrA. On the other hand, the PrrBc derivatives (PrrBc264 and PrrBc260) whose DDs were impaired by C-terminal deletion did not interact with PrrA. PrrB DD is a PrrB derivative that consists of 70 amino acids and contains only the DD of PrrB (Fig. 4A, C). The yeast strain (PrrA/PrrB DD) expressing both GAL4AD–PrrA and GAL4BD–PrrB DD fusion proteins grew on histidine-deficient medium. Taken together, the results suggest that the DD of PrrB is the region in which PrrB interacts with PrrA. Interestingly, the yeast strains expressing the PrrBc derivatives that contain the intact DD and impaired (or deleted) CA domain grew better on histidine-deficient medium supplemented with 3AT than the positive-control yeast strain (PrrA/PrrBc) on the same growth medium, indicating that the PrrBc derivatives lacking the intact CA domain interact with PrrA more strongly than does PrrBc. In good agreement with this observation, -galactosidase assay revealed that an approximately twofold higher activity of -galactosidase was detected in cell lysates of the yeast strain (PrrA/PrrBc270) as compared to the yeast strain (PrrA/PrrBc). This result suggests that the CA domain with PrrA exerts a negative effect on the PrrB interaction. Western blotting analysis was performed with a mAb against GAL4BD to detect the PrrBc derivatives fused to GAL4BD in yeast cells (Fig. 4B). All the PrrBc derivatives fused to GAL4BD as well as the GAL4BD–PrrBc fusion protein were detected immunologically at appropriate positions in the gel, indicating that the inability of the yeast strains (PrrA/PrrBc264 and PrrA/PrrBc260) to grow on histidine-deficient medium is not the result of instability of the fusion proteins in the yeast cell. The bands with molecular masses of 31 kDa probably resulted from non-specific cross-reactions of the antibody.
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). The yeast strain (PrrA/PrrB DD) expressing both GAL4AD–PrrA and GAL4BD–PrrB DD grew on histidine-deficient medium, while the yeast strain (DevR/PrrB DD) expressing the heterologous pair of the fusion proteins was unable to grow on the same medium (Fig. 5B). This result indicated that the DD of PrrB is sufficient in vivo to provide recognition specificity for its cognate response regulator, PrrA. In the case of the DevS histidine kinase, the DD of DevS (DevS DD) interacted with both DevR and PrrA, although interactions of DevS DD with its partner DevR appeared to be significantly stronger than those with PrrA. The control yeast strain (P/DevS DD) harbouring the empty prey vector did not grow on histidine-deficient medium (data not shown). The DevS derivative DevS DD+ATP, consisting of both the DD and CA domain, did not interact with PrrA, but gave interaction signals with its cognate response regulator, DevR. Taken together, the results shown in Fig. 5 suggest that the DD of the histidine kinase serves as the interaction region with its cognate response regulator and is sufficient to make both specific and non-specific interactions with its cognate response regulator and other related proteins. However, the presence of the CA domain masks the non-specific interactions (either directly or indirectly) and allows only for the specific interaction between a kinase and its cognate response regulator.
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-helix and turn within the PrrB DD are important for specific interactions between PrrB and PrrA-helix–turn–-helix (Tomomori et al., 1999). Our next question to be addressed was which portion of the DD plays a significant role in providing recognition specificity for its interaction with its cognate response regulator? To address this question, DNA fragments encoding the chimeric DDs (BS DD I and BS DD II) consisting of the -helices with two different origins (PrrB and DevS) were generated by recombinational PCR and cloned into the bait vector. The BS DD I polypeptide is composed of the first -helix (-helix 1) and turn derived from PrrB and the second -helix (-helix 2) from DevS (Fig. 6A). Protein–protein interactions between BS DD I and PrrA were observed in the yeast two-hybrid assay. In contrast, BS DD I did not interact with DevR (Fig. 6B). Another chimeric polypeptide BS DD II is made up of the -helix 1 from PrrB and the -helix 2 and turn from DevS. BS DD II did not interact with either PrrA or DevR. These results suggest that -helix 1 and the flanking turn structure of the PrrB DD determine the recognition specificity for the PrrA response regulator.
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, Western blotting analysis with a GAL4BD-specific antibody revealed that all the chimeric proteins fused to GAL4BD were normally expressed, indicating that the lack of interactions in the yeast two-hybrid assay observed for BS DD II is not due to instability of the chimeric proteins in the yeast two-hybrid system.
The EnvZ protein of E. coli is a histidine kinase that is involved in osmosensing and controls the phosphorylation state of the OmpR response regulator. The amino acid sequence of the EnvZ DD shows a high level of similarity with that of the PrrB DD, especially in the region encompassing -helix 1 and the turn (Fig. 7A). If the -helix 1 and turn of the DD are important for specific interactions between the DD and its cognate response regulator, the DD of EnvZ might be expected to interact not only with OmpR but also with PrrA. As shown in Fig. 7(B), the DD of EnvZ indeed interacted with PrrA as well as with its cognate response regulator, OmpR. The control yeast strain (P/EnvZ DD) harbouring the empty prey vector did not grow on histidine-deficient medium (data not shown). -Galactosidase assays were performed on cell-free lysates of the yeast strains as a measure of the strength of the protein–protein interactions. The interaction of the EnvZ DD with OmpR was shown to be much stronger than that with PrrA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2002) and to identify the histidine kinases which specifically interact with the DivK response regulator of Caulobacter crescentus (Ohta & Newton, 2003). While studying protein–protein interactions between the PrrB histidine kinase and the PrrA response regulator, we found that insertion of a linker of approximately 60 amino acids (including the linker of 32 amino acids provided by the prey vector itself) between GAL4AD and PrrA was required for the successful yeast two-hybrid assay. However, the GAL4AD–DevR fusion protein without the additional linker interacted with GAL4BD–DevS (data not shown), implying that the necessity of the additional linker between GAL4AD and response regulator depends on the type of response regulator.
Protein–protein interactions between the GAL4AD–PrrA and GAL4BD–PrrBc fusion proteins were clearly detected in the spotting assay, while those between GAL4AD–PrrBc and GAL4BD–PrrA were not (data not shown). This phenomenon has been observed elsewhere for the NtrBC two-component system and explained by instability of the GAL4AD–NtrB fusion protein in the yeast cell (Martinez-Argudo et al., 2002).
It was demonstrated in this study that the interaction surfaces of PrrA and PrrB reside in the regulatory domain and the DD, respectively, which is consistent with the fact that the autophosphorylated histidine in the DD must be closely juxtaposed to the conserved aspartate in the regulatory domain for the phosphotransfer reaction to ensue. Yeast two-hybrid studies on the NtrBC system have revealed that the 110 amino acid long NtrB fragment containing the DD specifically interacts with the regulatory domain of NtrC (Martinez-Argudo et al., 2002). In this study we further delineated the PrrB region with which PrrA interacts, and clearly showed that the PrrB DD consisting of only 70 amino acids interacted with PrrA and that impairment of the DD by further deletions abolished the interaction between PrrB and PrrA.
It has been suggested by structural (crystallographic and NMR titration) studies on the Spo0B-Spo0F and EnvZ-OmpR systems that the DDs of histidine kinases other than the Hpt (histidine-containing phosphotransfer)-containing CheA proteins are the sites with which their cognate response regulators interact (Tomomori et al., 1999; Zapf et al., 2000).
D63A and K113A mutations in the regulatory domain of PrrA abolished protein–protein interactions between PrrA and PrrB, suggesting that Asp-63 and Lys-113 among the active site residues of the PrrA regulatory domain are important not only in the function of PrrA but also for protein–protein interactions between PrrA and PrrB. Asp-63 of PrrA has been demonstrated elsewhere to be the amino acid that receives the phosphoryl group from PrrB (Comolli et al., 2002). Although there has been no report regarding the role of Lys-113 in PrrA function, it can be assumed from the earlier study on CheY that Lys-113 might be involved in a phosphorylation-induced conformational change in PrrA (Lukat et al., 1991). X-ray crystallographic studies on the D57A mutant form of CheY, which corresponds to the D63A form of PrrA, have revealed that the D57A mutation leads to a repositioning of the Lys-109 that is the counterpart of Lys-113 of PrrA and the 
-amino group of which is in close proximity to the Asp-57 -carboxylate group (Sola et al., 2000). From this observation, together with our result, we assume that the precise positioning of Asp-63 and Lys-113 within the active site of PrrA is important in maintaining the correct conformation of PrrA that is required for its interaction with PrrB. The T91A mutation of PrrA did not affect protein–protein interactions between PrrA and PrrB, which is in good agreement with the earlier finding that the T87A mutant form of CheY is phosphorylated normally by phosphorylated CheA (Appleby & Bourret, 1998), although this mutation renders CheY non-functional. Asp-19 and Asp-20 of PrrA are predicted to be involved in coordination of the catalytically essential Mg2+ ion together with Asp-63 (Lukat et al., 1990). Mutations of the corresponding amino acids to alanine or asparagine have been reported to convert the PhoB and OmpR response regulators into non-phosphorylatable forms (Brissette et al., 1991; Zundel et al., 1998). The D19A and D20A mutations of PrrA did not affect the protein–protein interactions between PrrA and PrrB, indicating that the mutations did not alter PrrA conformation to an extent sufficient to abolish its interaction with PrrB.
We showed here that the recognition specificity of a histidine kinase for its cognate response regulator is conferred primarily by its DD. Using the chimeric DDs it can be inferred that -helix 1 and its neighbouring turn structure are more important in conferring the recognition specificity on the DD. Although -helix 1 of the DD shows some sequence conservation around the conserved histidine, the amino acid sequence of -helix 1 and its neighbouring turn region appears to be sufficiently variable to provide the recognition specificity when the primary structures of different histidine kinases are compared (Grebe & Stock, 1999). An NMR titration experiment has suggested that OmpR interacts with the four-helix bundle of EnvZ most strongly at the lower part that corresponds to the C-terminal region of -helix 1, turn, and N-terminal region of -helix 2 (Tomomori et al., 1999). On the basis of the results presented here as well as those reported elsewhere (Tomomori et al., 1999; Martinez-Argudo et al., 2002; Ohta & Newton, 2003), we assume that the lower part of the four-helix bundle within the histidine kinase serves as a docking site for its cognate response regulator and that -helix 1 and its neighbouring turn of the DD provide to some extent the recognition specificity for its response regulator.
The CA domain of the histidine kinase is known to catalyse the phosphotransfer reaction from ATP to the conserved histidine residue within the DD (Park et al., 1998). As shown in Fig. 5(B), the DevS derivative containing both the DD and CA domain exhibited a higher recognition specificity for DevR than did the DevS derivative consisting of the DD alone, which interacted also with the non-cognate response regulator PrrA. This result clearly reveals that the CA domain of the histidine kinase provides additional recognition specificity to the histidine kinase in case the DD of the histidine kinase alone does not provide sufficient recognition specificity for its interaction with the response regulator. The specificity for protein–protein interactions between a histidine kinase and its response regulator appears to be provided not only by the DD that serves as an interaction surface, but also by the CA domain that catalyses the phosphotransfer reaction.
Deletion of the CA domain from PrrBc led to an increase in the interaction affinity of PrrBc for PrrA. This observation can be explained by the following: (i) the CA domain provides recognition specificity additional to that of the DD, which reduces the strength of protein–protein interactions between PrrBc and PrrA, or (ii) the GAL4BD–PrrBc fusion protein might phosphorylate the GAL4AD–PrrA fusion protein in the yeast cell. Phosphorylation might reduce the affinity of PrrA for PrrB. Earlier, it has been reported that the affinity of the phosphorylated OmpR for EnvZ is weaker than that of the non-phosphorylated OmpR (Mattison & Kenney, 2002).
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Received 2 March 2006;
revised 19 April 2006;
accepted 27 April 2006.
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