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Originally published In Press as doi:10.1074/jbc.M303042200 on April 17, 2003

J. Biol. Chem., Vol. 278, Issue 26, 23579-23585, June 27, 2003
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The H Box-harboring Domain Is Key to the Function of the Salmonella enterica PhoQ Mg2+-sensor in the Recognition of Its Partner PhoP*

María E. Castelli {ddagger} §, Ana Cauerhff ¶ ||, Marcela Amongero {ddagger}, Fernando C. Soncini {ddagger} || ** and Eleonora García Véscovi {ddagger} || {ddagger}{ddagger}

From the {ddagger}Instituto de Biología Molecular y Celular de Rosario (Consejo Nacional de Investigaciones Científicas y Técnicas), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario and Laboratorio de Inmunología Estructural-Fundación Instituto Leloir, Universidad de Buenos Aires-Consejo Nacional de Investigaciones Científicas y Técnicas, S2002-LRK Rosario, Argentina

Received for publication, March 25, 2003 , and in revised form, April 16, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In two-component signaling systems, the transduction strategy relies on a conserved His-Asp phosphoryl exchange between the sensor histidine kinase and its cognate response-regulator, and structural and functional consensus motifs are found when comparing either the diverse histidine kinases or response regulators present in a single cell. Therefore, the mechanism that guarantees the specific recognition between partners of an individual pair is essential to unequivocally generate the appropriate adaptive response. Based on sequence alignments with other histidine kinases, we dissected the Salmonella enterica Mg2+-sensor PhoQ in different subdomains and examined by in vivo and in vitro assays its interaction with the associated response regulator PhoP. This signal transduction system allows Salmonella to withstand environmental Mg2+ limitation by triggering gene expression that is vital throughout the infective cycle in the host. Using resonant mirror biosensor technology, we calculated the kinetic and equilibrium binding constants and determined that the His-phosphotransfer domain is essential for the PhoQ specific recognition and interaction with PhoP. Additionally, we show the role of this domain in the bimolecular transphosphorylation and provide evidence that this region undergoes dimerization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Two-component systems, either orthodox or multistep phosphorelays, have been largely demonstrated to be the most widespread signal transduction mechanism in eubacteria, and a wealth of these systems have been examined by genomic homology analysis, genetic mutation screening, and partial biochemical characterization. Members of this family of proteins are also present in eukaryotes such as yeast, fungi, and plants (reviewed in Ref. 1). These systems play a central role in bacterial adaptation to extracellular conditions, acting as communication channels between the environment and the inner cellular functions.

Although there are a variety of modular arrays in the two-component system family of proteins, in essence, they consist of a sensor protein, typically a histidine kinase (HK)1 responsible for the detection of a specific environmental signal that, through a His-Asp phosphotransfer mechanism, defines the activity state of its associated response regulator (RR). This RR ultimately controls the generation of an adequate adaptive response, most frequently by modulating gene expression (2, 3). The growing information that proceeds from the analysis of the entire genomic sequences of different bacteria clearly shows that a single cell can harbor 30–60 distinct His-Asp-related phosphotransfer signal transduction pairs (4–7). In light of the complex network of homologous systems that populate a single bacterial cell, one critical and yet not well understood aspect of the mechanism is the basis for the specificity of the recognition between the HK and its cognate RR. Therefore, the characterization of the interaction between the two components is basic to understand the phosphotransfer reactions and to determine the essence for the fidelity in the recognition throughout the signaling process.

Two broad types of HK have been defined in terms of the relative location of their substrate His and surrounding residues (H box) and the ATP catalytic binding domain: class I and class II (8). In class I, the prevalent type among bacterial HK classically represented by the osmosensor EnvZ, the H box is immediately followed by the ATP-binding domain. In the vast majority of the orthodox systems in which the sensor protein belongs to class I, this protein is a transmembrane receptor (9). In the type II enzymes (whose only known member is the chemotaxis HK CheA), the H box is located in the P1 domain at the N terminus (10, 11). NMR and x-ray diffraction studies generated detailed structural knowledge of a number of bacterial RR such as OmpR (12), CheY (13), NtrC (14), PhoB (15), NarL (16), and SpoOF (17). In parallel, structures of the catalytic or the His-harboring subdomains of HK that include EnvZ, PhoQ, ArcB, and CheA and a phosphorelay phosphotransferase, SpoOB, have also been elucidated (8, 18–20). However, in terms of partner interaction, detailed structural analysis has only been provided for the interaction between CheY and CheA from Escherichia coli (21–23), and SpoOF and SpoOB from Bacillus subtilis (5, 24, 25), where amino acidic residues involved in the mating have been disclosed. It is worth pointing out that, as mentioned above, CheA is an atypical HK, whereas SpoOB, component of the Bacillus sporulation phosphorelay cascade, is not an HK and consists in an isolated phosphotransferase-module that lacks the catalytic ATP-binding domain, although exceptionally retaining the capacity to form a four-helix bundle superimposable onto the homologous EnvZ domain (4). Collectively, from these studies it becomes apparent that recognition determinants on both partners of a HK-RR pair are essential for the specific interaction.

In the pathogenic bacteria Salmonella enterica serovar Typhimurium (Salmonella typhimurium), the PhoP/PhoQ two-component system governs the adaptation to Mg2+ limited-media (26–28). The detection of this parameter allows Salmonella to discern extracellular versus intracellular environments and to pleiotropically control the expression of factors that determine the successful invasion and spread in the host (reviewed in Refs. 29 and 30). PhoQ is a class I HK anchored to the bacterial inner membrane by two hydrophobic membrane-spanning domains that delimit a periplasmic sensor region. High resolution structural information of PhoQ has only come out for E. coli PhoQ, where Marina et al. (19) described the crystal structure of the C-terminal catalytic region of the sensor that binds ATP. Additionally, previous work provided evidence about the Mg2+ and Ca2+ binding capacity of the S. typhimurium (28), E. coli (31), and Pseudomonas aeruginosa (32) PhoQ sensor domains. We recently showed that S. typhimurium PhoQ is a bifunctional sensor, demonstrating that the conformational change triggered by Mg2+ upon interaction promotes a switch in the activity of the sensor from a kinase-dominant to a phosphatase-dominant state (33). This was later reinforced by results obtained by other groups (34, 35). However, no studies have yet examined the molecular basis for the PhoP-PhoQ specific recognition in the transduction process cascade.

In this work we characterized the interaction between the cytoplasmic region of PhoQ and its partner PhoP using different in vivo (interference phenotypes expressing liberated sensor domains) and in vitro (affinity retention columns and resonant mirror biosensor technology) approaches. We demonstrate that the PhoQ His-phosphotransfer domain (also termed DHp, for dimerization histidine phosphotransfer, in other two-component systems (Ref. 10)) is responsible for the specific recognition and interaction with PhoP. Additionally, we provide evidence that this region undergoes dimerization.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents—Nitrocellulose membranes were from Amersham Biosciences. [{gamma}-32P]ATP and [{alpha}-32P]ATP (3000 Ci/mmol) were obtained from PerkinElmer Life Sciences. The oligonucleotides were purchased from Bio-Synthesis, Inc. (Lewisville, TX.). Cell culture media reagents were from Difco, and chemicals were from Sigma.

Strains, Plasmids, and Growth Conditions—The PhoQ-derived truncated proteins were generated by the polymerase chain reaction using forward primers PhoQDHp-CTF (5'-CGGGATCCGATGAGGCCTATCGAG-3'), PhoQ-CTF(C) (5'-GAGGATCCCATATGCTGCTCAAAAGCGAG-3'), PhoQ-CTF(D) (5'-GAGGATCCCATATGCTGGAAGAGATC-3'), each paired to the reverse primer QREV (5'-TATAAAGCTTATTCCTCTTTCTGTGTGGG-3); PhoQDHp-CTF (5'-CGGGATCCGATGAGGCCTATCGAG-3'), paired to QA-Ino-R2 (5'-TCCAAGCTTAACTGGCGCGATGCAG-3'); and EnvZCyt-CTF (5'-CGGGATCCGATGCCGTTAGTGGATCTT-3'), paired to EnvZCyt-REV (5'-ACGACAAAAGAGGCATAAGCTTGGG-3') and cloned between the BamHI and HindIII sites of plasmid pUHE21–2lacIq (36) to generate plasmids pUHE::phoQLCyt, pUHE::phoQCyt, pUHE::phoQCA, pUHE::phoQDHp, and pUHE::envZCyt, respectively. Construction of plasmids pUHE::phoQ and pUHE::phoQH277V has been described previously (33). All pUHE21–2lacIq BamHI-HindIII inserts were excised, purified, and subcloned into cohesive sites of pT7-7 vector to generate plasmids pT7-7::phoQLCyt, pT7-7::phoQCyt, and pT7-7::phoQCA, or into cohesive sites of pQE-30 or pQE-31 vectors (Qiagen), depending on the appropriate reading frame, to generate plasmids pQE-30::phoQLCyt, pQE-30::phoQCyt, pQE-30::phoQCA, pQE-31::phoQDHp, pQE-31::envZCyt, harboring N-terminal His6 tag fusion constructs. The PCR fragment obtained using PhoQ-CTF(C) and QREV primers with pUHE::phoQH277V as template was cloned between the BamHI and HindIII sites of plasmid pQE-30 to generate pQE-30::phoQCytH277V.

S. typhimurium strain 14028s pcgG9283::MudJ (27) was transformed with pUHE-derived constructs or the vector as control, and used for the interference assays.

E. coli BL21[DE3] strain transformed with pT7-7-derived constructs or the vector as control was used for the expression of the PhoQ-derived proteins and subsequent cell fractionation (33). The soluble cell fractions were used for the affinity retention assays described below. E. coli M15/pREP4 strain was transformed with pQE-30 or pQE-31-derived constructs to express and affinity-purify the N-terminal His-tagged PhoQ- or EnvZ-derived proteins for the biosensor experiments.

To express and purify PhoP-H6 protein, E. coli BL21[DE3] carrying plasmid pPB1020 was used. pPB1020 harbors a phoP His tag fusion gene (full-length phoP fused to 6 His codons in its C terminus) under the control of the T7{phi}10 promoter of the pT7-7 plasmid as described (33).

{beta}-Galactosidase Activity Assays—For the {beta}-galactosidase activity assays, bacteria were grown overnight with shaking at 37 °C in Luria-Bertani medium with addition of different concentrations of IPTG as indicated. Ampicillin was used at 50 µg/ml final concentration in the bacterial growth media when appropriate. {beta}-Galactosidase was determined in overnight cultures as described (37). Soluble or membrane protein extracts from these cultures were obtained by cell fractionation as described below, analyzed by Tricine-SDS-PAGE (16.5% polyacrylamide) (38) transferred to nitrocellulose and developed by Western blot with rabbit anti-PhoQ (C-terminal cytoplasmic domain) polyclonal antibodies.

Genetic and Molecular Biology Techniques—Plasmid DNA was introduced into bacterial strains by electroporation using a Bio-Rad apparatus as recommended by the manufacturer. Recombinant DNA techniques were performed according to standard protocols (39). PCR-derived constructions were confirmed by DNA sequence analysis performed using the fmol DNA sequencing system as recommended by the manufacturer (Promega Corp.).

Protein Purification and Cell Fractionation Protocols—E. coli BL21[DE3] strains transformed with pT7-7-derived plasmids were used to obtain soluble protein extracts containing overexpressed PhoQ-derived truncated proteins for the interaction assays in affinity retention columns. Briefly, overnight cultures were used to inoculate LB media containing 50 µg/ml ampicillin. They were then grown at 37 °C to logarithmic phase (optical density of 0.6), and induced by addition of 1.0 mM IPTG, for an additional 3 h with shaking. Cells were collected, washed once, and resuspended in a solution containing 25 mM Tris-HCl (pH 8.0), 50 mM KCl (TK buffer). Cells were subjected to sonication and centrifuged 30 min at 20,000 x g, and the supernatant was collected. Protein concentration was determined by the bicinchoninic acid assay (Bio-Rad), using bovine serum albumin as standard. These supernatants were diluted to a final concentration of 2.0 mg/ml. All procedures were carried out at 4 °C. The protein profile was determined by SDS-polyacrylamide gel electrophoresis (PAGE) analysis. All His6-tagged fusion proteins were purified as previously described (33).

Affinity Retention Columns—Soluble protein extracts obtained by cell fractionation of E. coli BL21[DE3] strains harboring the vector pT7-7 or overexpressing PhoQLCyt, PhoQCyt, or PhoQCA (400 µg of total protein) were pre-incubated with purified PhoP-H6 (100 µg of protein) in buffer TK, 30 min on ice. Each protein mixture was loaded onto a Ni2+-NTA resin column (0.3 ml), previously equilibrated with buffer TK and blocked with control soluble protein extract (600 µg of total protein) to avoid nonspecific binding. Columns were washed with 3.0 ml of buffer TK, followed by 3.0 ml of buffer TK with 0.1 M imidazole, and finally the bound proteins were eluted with 150 µl of buffer TK with 1.0 M imidazole. Control columns were performed with each individual protein fraction and treated following the same sequential steps described above. An aliquot of 20 µl of each final elution fraction was analyzed by SDS-PAGE (12% polyacrylamide), transferred to nitrocellulose, and developed by Western blot with rabbit anti-PhoQCyt or anti-PhoP-H6 polyclonal antibodies.

All Western blots were probed with the first rabbit polyclonal antibody, as indicated, and developed by incubation with protein A conjugated with phosphatase, coupled to a chromogenic reaction using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates.

In Vitro Phosphorylation and Phosphotransfer Assays—For the autokinase assay, His-tagged affinity-purified proteins (2.0 µg of total protein) were incubated with 50 µM [{gamma}-32P]ATP (7500 cpm/pmol, PerkinElmer Life Sciences), 10 min in a 30-µl reaction mixture containing buffer TKM (25 mM Tris-HCl, 50 mM KCl, 1 mM MgCl2). The phosphotransfer assay was carried out by co-incubating 2.0 µg of PhoP and 2.0 µg of PhoQCyt with [{gamma}-32P]ATP in a 30-µl volume for 10 min, using the same reaction media described for the autokinase activity. For the transphosphorylation reactions, 2.0 µg of each individual protein (PhoQCyt, PhoQCA, PhoQDHp, or PhoQCytH277V) or the indicated combination of two proteins, were incubated in a 30-µl reaction mixture for 20 min, using the same reaction media described for the autokinase activity. All reactions were carried out at 37 °C. The reactions were stopped by adding 5x SDS-PAGE loading buffer (2.5% {beta}-mercaptoethanol, 9% glycerol, 10% SDS, 600 mM Tris-HCl (pH 6.8), 0.006% bromphenol blue). All reactions were analyzed by Tricine-SDS-PAGE (16.5% polyacrylamide), transferred to nitrocellulose, and then subjected to autoradiography.

Direct Photoaffinity Labeling—The His-tagged PhoQ-derivatives PhoQCyt and PhoQCA were photoaffinity-labeled with [{alpha}-32P]ATP using a method previously described (40). The reaction mixtures (40 µl) contained each purified protein in a concentration of 0.3 µg/µl in buffer TKM with 5% glycerol, and 20 µCi of [{alpha}-32P]ATP. Reaction mixtures were incubated for 15 min on ice in the dark; after this period 20 µl were removed and exposed to UV light for 5 min, on ice. UV light was generated by an ultraviolet lamp (model UVGL-55, UVP Inc.) at a distance of 5 cm from the sample. Treated and control samples were then subjected to Tricine-SDS-PAGE (16.5% polyacrylamide) followed by autoradiography.

Measurements of Equilibrium and Kinetic Constants—IAsys affinity sensor analysis experiments were conducted on the IAsys Plus apparatus (Affinity Sensors Ltd., Saxon Hill, Cambridge, UK). Purified proteins were covalently coupled to carboxymethyl dextran sensor chips (Affinity Sensors Ltd.) via free amino groups using N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)carboxidiimide coupling chemistry at 22 °C, using the protocol recommended by the manufacturer. His-tagged PhoP or His-tagged PhoQCyt were coupled to individual cuvettes giving a signal of 520 and 390 arc s, corresponding to 10.5 or 7.8 ng of protein, respectively. All binding reactions were carried out in buffer TKM plus 0.05% Tween 20, at 25 °C, with constant stirring set up at 95%, and the data were collected at interval of 0.3 s. Ligate binding to immobilized ligand was monitored at multiple ligate concentrations, ranging 10-fold below to at least 10-fold above preliminary estimates of equilibrium dissociation constants (KD) for each reaction. Kinetic analysis was performed using the FASTfit software (Affinity Sensors Ltd.), which provided the values of kon and kd at each ligate concentration. Second-order association rate constants (ka) were then obtained from a linear regression plot of kon versus ligate concentration. Dissociation equilibrium constants (KD) were then derived for each reaction from the equation: KD = kd/ka. For the PhoQCyt-chip interaction, constants were obtained from equilibrium data. Binding curves were performed at several concentrations of PhoQCyt or PhoQDHp (10-fold below to at least 100-fold above preliminary estimates of KD), and the ligate was allowed to bind for 30 min to obtain a saturating equilibrium response (Req). Bovine serum albumin (1.0 µM) interaction was tested as a non-related control protein when using either PhoQCyt- or PhoP-chip, rendering basal level sensorgrams.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PhoQ-derived Proteins That Retain the DHp Domain Interfere with the PhoP/PhoQ Transduction Cascade—Domain liberation has proved to be a useful in vivo tool when analyzing the functional architecture of multidomain proteins such as the histidine kinases CheA and UhpB (41–43). This approach is based on the inhibitory effect that a subcloned and overexpressed protein domain exerts when it interferes by competition with the interplay between the endogenously expressed partner proteins, provided an interaction takes place between them. Taking advantage of this approach, we used truncated proteins derived from the cytoplasmic domain of PhoQ to perform an in vivo screening for the protein subdomain that retained the capacity to interfere with the signaling cascade and thus to interact with its constituents. As is depicted in Fig. 1A, PhoQLCyt spans the whole cytoplasmic region encompassing the putative linker (L), the H box-harboring region (DHp), and the catalytic ATP-binding domain (CA), PhoQCyt lacks the putative L domain, PhoQDHp contains the H box-harboring domain but lacks CA, and PhoQCA harbors only the CA domain. All constructs were cloned into vector pUHE21–2lacIq and each resulting plasmid was introduced into the S. typhimurium 14028s pcgG9283::MudJ strain by electroporation. Protein expression was induced by addition of increasing concentrations of IPTG to the growth media as indicated in Fig. 1B. The expression levels of the reporter gene pcgG, a representative phoP-activated gene, was followed by measuring the {beta}-galactosidase activity from the pcgG-lacZ transcriptional fusion. As shown in Fig. 1B, intact PhoQ, mutant PhoQH277V, PhoQLCyt, PhoQCyt, and PhoQDHp showed a 3.3-, 3.3-, 2.2-, 4.0-, and 2.6-fold inhibition of pcgG expression, respectively (calculated at 0.3 mM IPTG induction), although PhoQCA showed no effect on the reporter gene expression when compared with the strain that harbors the control vector. Cellular fractionation followed by immunodetection analysis showed that, with the exception of membrane-anchored PhoQ and PhoQH277V, all PhoQ-derived proteins expressed partitioned into the soluble cell fraction (Fig. 1C). In the immunodetection assay PhoQDHp appears as a faint band, although in Coomassie Blue-stained gels it showed the same intensity as the other truncated proteins, indicating that this protein did not react efficiently with the rabbit anti-PhoQCyt polyclonal serum used to develop the Western (this was also the case when we immunodetected a His-tagged Pho-QDHp purified protein with this antibodies; data not shown). These results suggested that all PhoQ-derived proteins that retain the DHp domain were able to interfere with the transduction cascade that leads to gene activation. This interference effect could conceivably be attributed to different mechanisms that can simultaneously take place: (a) the dimerization or oligomerization of the endogenous PhoQ with the overexpressed proteins rendering non-functional complexes, (b) the sequestration of the chromosomally expressed PhoP by the overexpressed full-length PhoQ or its truncated derivatives, and (c) a phosphatase activity acting on phospho-PhoP. Although we cannot completely rule out the third possibility, we favor the interaction/sequestration alternative as being the dominant cause for down-regulation because: (a) when full-length PhoQ is overexpressed pcgG inhibition is observed even when cells were grown in low Mg2+, a condition that turns down the phosphatase activity of PhoQ, and (b) when we expressed the mutant PhoQH277V protein, which lacks the phospho-PhoP phosphatase activity (33), the inhibition of pcgG expression was almost identical to the one obtained for full-length PhoQ (Fig. 1B).



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  		FIG. 1.
In vivo interference assay using PhoQ-truncated derivatives. A, schematic representation of the PhoQ sensor predicted domains and derived truncated proteins. T1 and T2 indicate the first and second transmembrane domains, SD, LD, DHp, and CA correspond to the sensor, linker, dimerization histidine-phosphotransfer, and ATP-binding catalytic domains, respectively. H denotes the position of the His-277 residue in the DHp domain. The N- and C-terminal amino acid residue positions of each PhoQ-derived protein relative to the entire PhoQ are shown above ends of each bar. B, transcription of the pcgG gene from S. typhimurium pcgG::MudJ strain harboring plasmids pUHE (vector), pUHE::phoQ (PhoQ), pUHE::phoQH277V (PhoQH277V), pUHE::phoQLCyt (PhoQLCyt), pUHE::phoQCyt (PhoQCyt), pUHE::phoQCA (PhoQCA), pUHE::phoQDHp (PhoQDHp), or pUHE::envZCyt (EnvZCyt). Cells were grown overnight in LB medium with the indicated concentration of IPTG. {beta}-Galactosidase activity from the pcgG-lacZ transcriptional fusion was measured as described under "Experimental Procedures." Results are average of three independent assays, and error bars correspond to standard deviation. C, membrane (PhoQ and PhoQH277V) or soluble (PhoQLcyt, PhoQCyt, PhoQCA, PhoQDHp) cell fractions prepared from aliquots withdrawn from the expression assay (0.3 mM IPTG) shown in B were separated by Tricine-SDS-PAGE (16% polyacrylamide; 50 µg of total protein/well) and analyzed by Western blot developed using anti-PhoQ (C-terminal cytoplasmic domain) polyclonal antibodies.

 

PhoQ-derived Proteins Encompassing the DHp Domain Interact with PhoP in an Affinity Retention Assay—To concentrate in the analysis of the PhoP-PhoQ interaction, we examined the capacity of the soluble PhoQ-derived proteins to interact with PhoP. With this purpose we performed an in vitro binding assay using purified His-tagged-PhoP pre-incubated with soluble bacterial extracts containing truncated PhoQ-derived proteins (Fig. 2A). To improve protein yields, phoQ-derived constructs were transferred and expressed under the control of the T7 {phi}10 promoter to the pT7-7 vector in E. coli BL21[DE3] and cell fractionation was performed as described under "Experimental Procedures." As is shown in Fig. 2B, the immunodetection analysis of the proteins that co-eluted with PhoP (detected also by Western blot; Fig. 3B) from Ni2+-NTA resin with imidazole showed that both PhoQLCyt and PhoQCyt, but not PhoQCA, were affinity-trapped with PhoP-H6. Controls were performed to exclude the possibility that the different truncated PhoQ-derived proteins interacted nonspecifically with the column matrix; although PhoQCyt and PhoQCA alone did not show nonspecific binding to the Ni2+-NTA resin, Pho-QLCyt showed a low nonspecific binding, which accounted for ~5% of the eluted protein, being the remnant 95% co-eluted specifically with PhoP-H6. PhoQCytH277V was also tested and rendered an interaction behavior identical to the wild type PhoQCyt (data not shown). These results qualitatively indicated that the PhoQ derivatives that retain the H box-harboring domain exhibit the ability to interact with PhoP.



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  		FIG. 2.
PhoP-PhoQ interaction analysis by affinity retention chromatography. A, Coomassie Brilliant Blue-stained SDS-PAGE (12% acrylamide) showing the protein pattern that corresponds to the soluble extracts (50 µg of total protein/well) from strain E. coli BL21[DE3] harboring pT7-7 (vector), pT7-7::phoQLCyt (PhoQLCyt), pT7-7::phoQCyt (PhoQCyt), or pT7-7::phoQCA (PhoQCA), respectively, that were subsequently co-incubated with purified His-tagged PhoP. MWS, protein broad range molecular mass standards (New England Biolabs). B and C, protein extracts as shown in A were pre-incubated with a fixed amount of purified PhoP-H6, loaded onto Ni2+-NTA-agarose columns, and treated as described under "Experimental Procedures." The PhoP-H6 affinity-trapped proteins that co-eluted with imidazole were analyzed by SDS-PAGE (12% polyacrylamide) followed by transfer to nitrocellulose and developed using rabbit anti-PhoQ (C-terminal cytoplasmic domain) (B) or anti-PhoP polyclonal antibodies (C). The position of each protein is indicated by an arrow.

 


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  		FIG. 3.
In vitro activities of the purified PhoQ-derived truncated proteins. A, purified PhoQCyt or EnvZCyt (2.0 µg of each protein, in a 30-µl reaction mixture) were individually incubated to allow for the autophosphorylation reaction. Asterisk (*) shows the positions of homodimeric phosphorylated PhoQCyt and EnvZCyt proteins. B, purified PhoQCyt was incubated alone or together with PhoP (2.0 µg of each protein, in a 30-µl reaction mixture) to allow for the phosphotransfer reaction. C, purified PhoQCyt, PhoQCA, or PhoQCytH277V were incubated alone or in the presence of PhoQDHp (2.0 µg of each protein, in a 30-µl reaction mixture) to allow for transphosphorylation. PhoQDHp alone was incubated in the same reaction medium as control. Reactions were carried out for 10 min (A and B) or 20 min (C) at 37 °C, in buffer TKM containing 50 µM [{gamma}-32P]ATP (7500 cpm/pmol), as described under "Experimental Procedures." Samples were separated by Tricine-SDS-PAGE (16.5% polyacrylamide) and transferred to nitrocellulose, followed by autoradiography. D, purified PhoQCyt or PhoQCA (0.3 µg/µl each, in a 40-µl reaction mixture) were individually incubated for 15 min at 4 °C, in buffer TKM containing 20 µCi of [{alpha}-32P]ATP. The reaction mixture was split, and half of the total volume was exposed 5 min to UV light, whereas the rest was incubated in the dark as described under "Experimental Procedures". Samples were separated by Tricine-SDS-PAGE (16.5% polyacrylamide) and transferred to nitrocellulose, followed by autoradiography.

 

The PhoQ-derived Proteins Display Intact Functional Properties—Before performing biosensor assays, we examined whether the different PhoQ-derived proteins exhibited their putative biochemical activities. Our previous work demonstrated the autokinase and Mg2+-modulated phosphatase activities of the intact Salmonella enterica PhoQ. However, there was no biochemical demonstration that, as was shown for other sensors such as EnvZ (44), PhoR (45), or NtrB (46), PhoQ could be dissected in subdomains able to individually exert catalytic activities and reconstitute the functional protein when mixed. PhoQ-derived proteins were expressed as N-terminal His6-tagged fusions and affinity-purified using Ni2+-NTA columns, as described under "Experimental Procedures." Because the His-tagged PhoQLCyt protein could not be efficiently expressed and purified, and in the previous experiments it had not revealed significant differences with PhoQCyt, it was not used in subsequent analysis. Fig. 3 shows the resultant autoradiograms from the autophosphorylation reactions of purified Pho-QCyt, and EnvZCyt (Fig. 3A), the PhoQCyt -> PhoP phosphotransfer (Fig. 3B), and the transphosphorylation from PhoQCyt to the PhoQDHp domain (Fig. 3C). When either PhoQCA or PhoQCytH277V were incubated with PhoQDHp, phosphorylation occurred in this last domain, whereas PhoQCA, PhoQDHp, or PhoQCytH277V did not autophosphorylate (Fig. 3C). Additionally, taking into account that the PhoQCA domain harbors the conserved motifs that bind ATP leading to autophosphorylation of the sensor, its ability to bind [{alpha}-32P]ATP by UV cross-linking was assayed. Fig. 3D shows that PhoQCA (and also PhoQCyt, used as control) retained the capacity to bind the nucleotide. Interestingly, bands that corresponded to the predicted PhoQCyt and EnvZCyt homodimers could be visualized in the autoradiograms obtained from the autokinase assay (Fig. 3A; the identity of the bands was confirmed by immunodetection; data not shown). Together, these results show that the PhoQ-derived proteins used in this work are biochemically functional.

Quantification of the Strength of the PhoP-PhoQ Interaction—To accomplish a detailed characterization of the PhoP-PhoQ interaction observed, we used resonant mirror biosensor technology that allows to quantify the strength of protein-protein interactions. In this approach, real-time binding of a ligate to an immobilized ligand covalently attached to the dextran matrix of a sensor chip is monitored as a change in refractive index occurring at the surface of the biological layer and is directly proportional to the amount of bound protein (47). To carry out the biosensor assays, purified PhoP was first attached to the sensor chip and its interaction with the PhoQ-derived proteins was tested. Simple bimolecular interactions adequately described our data in all cases (using FASTfit program from IAsys; see "Experimental Procedures"). Table I shows association (ka) and dissociation (kd) rate constants, and dissociation equilibrium constant (KD) obtained for each experiment assuming a pseudo-first-order kinetic behavior. Increasing concentrations of PhoQCyt were tested in the PhoP cuvette (Table I, immobilized PhoP). From this data, a dissociation equilibrium constant (KD) for the PhoP-PhoQCyt interaction was estimated to be 1.19 x 10–7 M. Interestingly, when we tested PhoQDHp it retained the capacity to interact with PhoP, being its calculated KD = 7.94 x 10–7 M, whereas PhoQCA did not show appreciable interaction with PhoP (KD = 9.59 x 10–5 M). To assess the specificity of the obtained signals, we assayed PhoP interaction with EnvZCyt, the cytoplasmic region from the homologous S. typhimurium EnvZ sensor. The sensorgrams showed no detectable association of EnvZCyt with PhoP. In addition, PhoQCytH277V showed kinetic binding constants similar to the ones obtained with PhoQCyt.


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  		TABLE I
Kinetic analysis of the interaction between PhoP and PhoQ and the dimerization of PhoQ using IAsys biosensor technology

The kinetic and equilibrium parameters were determined as described under "Experimental Procedures." NI, no interaction parameters could be calculated. Errors shown are standard errors of the mean.

 

To perform counterpart experiments we immobilized Pho-QCyt to a new cuvette (Table I, immobilized PhoQ). In these assays the interaction with PhoP was not detected. Absence of interaction could be the result of steric hindrance of the interacting surface upon immobilization to the dextran matrix. However, we monitored the generation of the PhoQCyt-PhoQCyt complex when PhoQCyt was tested as ligate. The equilibrium interaction assay yielded a KD = 1.62 x 10–7 M. When PhoQDHp was used, interaction with PhoQCyt was also monitored, rendering a calculated KD = 4.14 x 10–7 M. In these experiments dissociation of either the PhoQCyt-PhoQCyt or PhoQCyt-Pho-QDHp complexes could be achieved by adding PhoP, and this dissociation was more effective than sole buffer addition (data not shown), indicating that PhoP could displace the PhoQCyt-PhoQCyt interaction.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the intracellular milieu, where an estimated 40 distinct types of two-component pairs are differentially regulated by diverse input signals, crucial specific interaction instances assure the generation of an accurate response: the recognition of the signaling ligand by the proper sensor (in this case Mg2+ binding to PhoQ), the pairing of the sensor to a unique matching response regulator (i.e. PhoQ to PhoP), and the binding of the response regulator to its specific cellular target (PhoP binding to regulatory regions in the DNA).

Previous work revealed different aspects of the Mg2+-PhoQ interaction such as the identification of the sensing dedicated domain and the characterization of the interaction with divalent cations (27, 28, 31, 35, 48). In this work we sued to identify the PhoQ domain that sustains the specific recognition of PhoP and examined the kinetic parameters that characterize the interaction.

In this work we showed by an in vivo domain liberation experiment that full-length PhoQ, the cytoplasmic derivatives PhoQLCyt and PhoQCyt, and the putative dimerization phosphotransfer-harboring domain PhoQDHp were able to down-regulate the expression of a PhoP-activated gene in inducing conditions (i.e. in Mg2+-limiting concentrations). In contrast, neither the isolated PhoQ catalytic domain (PhoQCA) nor EnvZCyt was able to exert a detectable effect, even though all the proteins tested were equally expressed. The potential involvement of the phospho-PhoP phosphatase activity in the inhibition was excluded because either overexpressed full-length PhoQ in its kinase-dominant state or a mutant PhoQ that lacks phosphatase activity (PhoQH277V) showed pcgG down-regulation to the same extent as the truncated proteins. Accordingly, in vitro retention of PhoQ-derived proteins by PhoP-bound affinity columns showed that the liberated C-terminal truncated proteins (PhoQLCyt and PhoQCyt, but not Pho-QCA) preserved the capacity, and thus the proper conformation, to effectively recognize and interact with PhoP with a binding strength that overcame subsequent column washes and allowed the elution of the complexes with imidazole. Therefore, these results pointed out that all PhoQ-derived constructs harboring the DHp region contained a relevant and specific interaction determinant.

Optical biosensor technology has proved to be an adequate biophysical tool for the analysis of both kinetic and equilibrium parameters to characterize a wide variety of biological meaningful interactions (49, 50). The analysis of the biosensor assays rendered a KD = 1.19 x 10–7 M for PhoP-PhoQCyt and a KD = 7.94 x 10–7 M for PhoP-PhoQDHp, whereas the KD for the PhoP-PhoQCA interaction was 2.9 or 2.1 orders of magnitude higher, respectively. These results clearly demonstrated that the DHp region is essential for the interaction and that the contribution of the CA domain, if any, is low. The lack of a detectable signal when using EnvZCyt showed that this biophysical approach is highly sensitive to the specificity of the measured interaction. The PhoQ-PhoP calculated dissociation equilibrium constant is in agreement to those obtained for the interaction of other two-component systems partners using biosensor technology, fluorescence anisotropy and polarization, or a kinetic simulation model, estimated as 4.25 x 10–7 M (51) or 1.96 x 10–6 M (52) for EnvZ/OmpR, 8.06 x 10–7 M for EvgS-O/EvgA (53), 3.0 x 10–8 M for VanS/VanR (54), and 3.0 x 10–8 M for CheA/CheY51YC (55). Here it is worth pointing out that differences in the estimated parameters could reflect structural differences among systems as well as divergences in the methodological approaches used.

Lately, efforts have been posed to elucidate how interaction is affected by the phosphorylation state of the components. Li et al. (56) working with the CheY/CheA system, and Mattison and Kenney (51) analyzing the OmpR/EnvZ interaction, found that upon phosphorylation, the response regulators reduce their affinity for the cognate kinases by 5- to at least 10-fold, respectively. In contrast, Yoshida et al. (52) showed that the EnvZ-OmpR interaction can be outcompeted with the same strength either by phosphorylated or by unphosphorylated OmpR. To explore this controversial matter, we assayed PhoQCyt incubated in buffer TKM with 1 mM ATP, or with 1 mM ADP, previous to the addition to the PhoP-chip. Both assays yielded essentially the same KD as the control PhoP-PhoQCyt (see Table I). These results suggest that the phosphorylation state of the partners may not modify the strength of the interaction. However, additional experiments are being conducted to further analyze this observation because, even when the same conditions as those employed for phosphotransfer experiments were used, the phosphorylation status of neither PhoP nor PhoQCyt could be evaluated throughout the binding assay.

The PhoQCyt-PhoQCyt interaction detected either by the biosensor assay or by electrophoretic analysis, the measured Pho-QCyt-PhoQDHp binding, together with the observed PhoQCyt-> PhoQDHp transphosphorylation reaction, provides evidence that the PhoQ sensor dimerizes, consistent with the model that applies for other two-component systems (comprehensively reviewed by Dutta et al. (Ref. 10)). EnvZCyt did not interact with immobilized PhoQCyt, therefore showing that the bimolecular interaction between sensor molecules is also ruled by recognition specificity. Although no results were obtained with the PhoQCyt-chip when using PhoP as ligate, an effective dissociation of PhoQCyt-PhoQCyt or PhoQCyt-PhoQDHp complexes was obtained by the subsequent addition of PhoP. These results strongly suggest that the immobilization of PhoQCyt to the cell matrix occurred preferentially in an orientation that rendered the PhoP-binding site sterically unavailable. Presumably, once PhoQCyt dimerization had taken place, added PhoP acted as a sink of free PhoQCyt or PhoQDHp and the formation of these complexes displaced the primary interaction equilibrium.

In light of the interaction results examined here, it is tempting to rationalize that the in vivo interference effect observed was caused by simultaneous formation of complexes between the overexpressed proteins and the endogenous PhoQ, and sequestration of PhoP. This also suggests that interpretations of in vivo assays that use protein overexpression should be taken cautiously because they could be biased by interaction effects.

Additionally, activity assays provided evidence that relevant functional, and thus structural, features of the isolated PhoQ subdomains analyzed in this work are preserved. We also demonstrated that these subdomains are able to reconstitute distinctive PhoQ activities including bimolecular transphosphorylation as previously described for analogous systems (44, 57–59).

In conclusion, the results showed in this work reveal that DHp is the PhoQ domain that concentrates structural and functional determinants that are key to homodimerization, to specific PhoP recognition and to signal-induced phosphotransfer activities.


   FOOTNOTES
 
* This work was supported in part by grants from Agencia Nacional de Promoción Científica y Tecnológica (Argentina), Fundación Antorchas, the Third World Academy of Sciences (Trieste, Italy), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), and Ministerio de Salud de la República Argentina. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of a fellowship from CONICET. Back

|| Career investigator of CONICET. Back

** Member of the Rosario National University Research Council; International Research Scholar of the Howard Hughes Medical Institute. Back

{ddagger}{ddagger} To whom all correspondence should be addressed. Tel.: 54-341-4356369; Fax: 54-341-4390465; E-mail: pat-bact{at}citynet.net.ar.

1 The abbreviations used are: HK, histidine kinase; RR, catalytic ATP-binding domain; DHp, H box-harboring region; CA, catalytic ATP-binding domain; L, linker; IPTG, isopropyl-1-thio-{beta}-D-galactopyranoside; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Ni2+-NTA, nickel-nitrilotriacetic acid. Back



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