HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 21, 20596-20603, May 27, 2005

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 280/21/20596    most recent M502015200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kneuper, H. Articles by Unden, G. Search for Related Content PubMed PubMed Citation Articles by Kneuper, H. Articles by Unden, G. Social Bookmarking                      What's this?

The Nature of the Stimulus and of the Fumarate Binding Site of the Fumarate Sensor DcuS of Escherichia coli^*

Holger Kneuper, Ingo G. Janausch, Vinesh Vijayan, Markus Zweckstetter¶, Verena Bock, Christian Griesinger, and Gottfried Unden||

From the Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg-Universität Mainz, 55099 Mainz, Germany and Max Planck Institut für Biophysikalische Chemie, 37077 Göttingen, Germany

Received for publication, February 22, 2005 , and in revised form, March 21, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
DcuS is a membrane-associated sensory histidine kinase of Escherichia coli specific for C₄ -dicarboxylates. The nature of the stimulus and its structural prerequisites were determined by measuring the induction of DcuS-dependent dcuB'-'lacZ gene expression. C₄-dicarboxylates without or with substitutions at C2/C3 by hydrophilic (hydroxy, amino, or thiolate) groups stimulated gene expression in a similar way. When one carboxylate was replaced by sulfonate, methoxy, or nitro groups, only the latter (3-nitropropionate) was active. Thus, the ligand of DcuS has to carry two carboxylate or carboxylate/nitro groups 3.1–3.8 Å apart from each other. The effector concentrations for half-maximal induction of dcuB'-'lacZ expression were 2–3 mM for the C₄-dicarboxylates and 0.5 mM for 3-nitropropionate or D-tartrate. The periplasmic domain of DcuS contains a conserved cluster of positively charged or polar amino acid residues (Arg¹⁰⁷-X₂-His¹¹⁰-X₉-Phe¹²⁰-X₂₆-Arg¹⁴⁷-X-Phe¹⁴⁹) that were essential for fumarate-dependent transcriptional regulation. The presence of fumarate or D-tartrate caused sharpening of peaks or chemical shift changes in HSQC NMR spectra of the isolated C₄-dicarboylate binding domain. The amino acid residues responding to fumarate or D-tartrate were in the region comprising residues 89–150 and including the supposed binding site. DcuS(R147A) mutant with an inactivated binding site was isolated and reconstituted in liposomes. The protein showed the same (activation-independent) kinase activity as DcuS, but autophosphorylation of DcuS was no longer stimulated by C₄-dicarboxylates. Therefore, the R147A mutation affected signal perception and transfer to the kinase but not the kinase activity per se.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bacterial cells contain large numbers of sensors for monitoring changes in chemical and physical parameters. The most common type of sensory system is represented by two-component sensors consisting of sensory histidine kinases and response regulators (1, 2). The response regulators mostly control the expression of target genes by transcriptional activation or repression. Many sensor kinases have a transmembrane arrangement with the sensory domain in the periplasm and the kinase domain in the cytoplasm. The activity of the kinase domain typically is regulated by the sensory domain by binding of an effector molecule, resulting in autophosphorylation of the kinase at a conserved histidine residue and subsequent transfer of the phosphoryl group to the response regulator. For most of the 28 identified suggested histidine kinases of Esherichia coli (3), neither the exact nature of the stimulus nor the stimulus binding sites are known. One of the few His kinases characterized in this respect is the citrate sensor CitA of enteric bacteria. Stimulus and stimulus binding site of CitA are well characterized (4–6).

The DcuS¹-DcuR two-component system (C₄-dicarboxylate uptake) of E. coli, consisting of the membrane-associated His kinase DcuS and of the response regulator DcuR, has been identified as a sensory system for fumarate and other C₄-dicarboxylates (7–9). DcuS-DcuR stimulates the expression of genes encoding carriers and enzymes for the catabolism of externally supplied C₄-dicarboxylates. One of the main targets of the DcuS-DcuR system is the dcuB gene, which encodes the C₄-dicarboxylate carrier DcuB catalyzing fumarate/succinate antiport during fumarate respiration (10, 11). dcuB is transcriptionally activated by the phosphorylation of DcuR in the presence of C₄-dicarboxylates and by the oxygen sensor FNR during anaerobiosis (7, 8, 12–14).

As a member of the CitA family of sensor kinases, DcuS shares sequence similarity and domain organization with the citrate sensor CitA (7, 9, 15). DcuS is predicted to be a transmembraneous protein with the sensory domain exposed to the periplasm. The periplasmic domain of 140 amino acids is arranged between the two transmembraneous helices of DcuS. The structure of the periplasmic domain of DcuS in solution has been determined using NMR spectroscopy (16, 17). The sensory domain consists of a novel -fold with -helices close to the N- and C-terminal ends of the periplasmic domain. The binding domain shows similarity to the PAS (Per-Arnt-Sim) domain of the photoactive yellow protein PYP of Halorhodospira halophila (18). The cytoplasmic part of DcuS consists of a further PAS domain of unknown function and a transmitter domain with the predicted phosphorylation site. DcuS has been solubilized in detergent and functionally reconstituted in liposomes (12). In this system, kinase activity of DcuS responds to the presence of fumarate and other C₄-dicarboxylates (12).

Previously, it has been suggested that DcuS uses C₄-dicarboxylates as the stimuli (7, 8, 12). This is in contrast to the high specificity of the homologous His kinase CitA for citrate, isocitrate, and tricarballylate (4, 19). So far the structure of DcuS-PD could be solved only without bound C₄-dicarboxylates (17), which precludes direct identification of the effector binding site. Therefore, we aimed at an identification of the stimulus binding site by genetic, biochemical, and NMR spectroscopic methods. The studies showed that DcuS has a remarkably broad specificity for stimulus molecules and suggested a defined binding pocket for the C₄-dicarboxylates and the presence of distinct amino acid residues for C₄-dicarboxylate binding. By these properties, DcuS can be clearly differentiated from the citrate sensor CitA. In addition, the effect of mutations in the effector binding site on the activity of the kinase domain and of its sensitivity to stimulation was tested for a better understanding of the protein in signal perception and transfer.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Genetic Methods—Standard molecular genetic methods were used (20). Genomic DNA was isolated according to Chen and Kuo (21). Plasmids were isolated using kits. PCR products were purified with the QIAquik PCR purification kit (Qiagen). DNA was extracted from agarose gels with the QIAquik gel extraction kit (Qiagen).

E. coli strains were transformed via electroporation or after treatment with RbCl (22, 23). The dcuS gene including the promoter region from E. coli AN387 (24) genomic DNA was amplified using the hot start method with oligonucleotide primers reHindDcuS (5'-TCG GCG ATT AAG CTT CGC GAC C-3') and liXbaDcuS (5'TTT CAG GCT TTC ATC TAG ATC GGT ATG-3'). The product was cloned via the HindIII and XbaI sites into pET28a (Novagen). The resulting plasmid, pMW181, carried the dcuS gene and its complete promoter region and was used as the template for mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene). The mutations in DcuS, verified by DNA sequencing, were created by replacing the codon for an amino acid residue with the codon for alanine or another amino acid (Table I). The plasmids carrying the mutations were used for expression of the corresponding mutant proteins from the dcuS promoter.

Isolation of DcuS and DcuR—DcuS was isolated after solubilization with detergent (12). The DcuS(R147A) mutant protein was isolated in similar yields using the same procedure. DcuR and DcuR(D56N) were isolated following the procedure of Janausch et al. (12).

Reconstitution of DcuS in Liposomes—Purified DcuS and DcuS(R147A) were each reconstituted in E. coli phospholipids (Avanti%20Polar%20Lipids">Avanti Polar Lipids Inc.) as described for DcuS (12). The liposomes were destabilized by the addition of Triton X-100 (0.8% w/v). Isolated DcuS or DcuS(R147A) in elution buffer was added at a phospholipid:protein ratio >20:1 (mg/mg) and stirred gently for 10 min at 20 °C. Degassed Bio-Beads SM-2 (Bio-Rad) pretreated as described by Holloway (25) were added to remove the detergent (10 mg beads/mg Triton X-100). The suspension was incubated overnight at 4 °C. The supernatant was then incubated with fresh Bio-Beads (10 mg beads/mg Triton X-100) for 1 h at 20 °C. The supernatant was removed with a pipette, frozen in liquid N₂, and stored at –80 °C.

Phosphorylation of DcuS and Phosphoryl Transfer to DcuR— An 80-µl aliquot of the proteoliposome suspension was adjusted to 10 mM MgCl₂, 1 mM dithiothreitol, and 20 mM fumarate (or other carboxylic acids as indicated) and subjected to three cycles of rapid freezing in liquid N₂ and slow thawing at 20 °C (26). After the final thawing, the proteoliposomes were kept for 1 h at 20 °C. 2.5 µl of [-³³P]ATP (110 TBq/mmol) then was added to attain final concentrations of 0.1–100 µM ATP. Isolated DcuR or DcuR(D56N) was added to the suspension in 4-fold excess over DcuS. At the times indicated, 10-µl samples were mixed with 10 µl of SDS loading buffer and a volume corresponding to 2 µg of DcuS/lane was applied to an SDS-polyacrylamide gel. After electrophoresis, the gel was exposed to an imaging plate (Fuji BAS-MP2040) for measuring the radioactivity in the bands in a PhosphorImager (12).

NMR Studies on Isolated Periplasmic Domain of DcuS (DcuS-PD)— Wild-type DcuS-PD and the mutant forms, DcuS(F120M)-PD, DcuS(R147A)-PD, and DcuS(H110A)-PD, were dissolved in a NMR buffer at pH 7.0 containing 45 mM sodium/potassium phosphate, 10% D₂O, 200 mM NaCl, 0.01% NaN₃ 50 µM Pefabloc, 50 mM glycine, and 4.5 mM imidazole. Titrations with the effectors were conducted by the stepwise addition of properly buffered fumarate and tartrate solutions (pH 7.0 and up to 300 mM final concentration) to a sample of 1.2 mM periplasmic domain of DcuS. Two-dimensional ¹⁵N-¹H heteronuclear single quantum coherence (HSQC) experiments were recorded for each step of the titration. Experiments were performed on Bruker DRX 800 and 600 MHz NMR spectrometers at 30 °C. The data were processed using NMRPIPE (27) and analyzed using SPARKY.²

Expression Studies with dcuB'-'lacZ—E. coli IMW237 (MC4100 (dcuB'-'lacZ) grown anaerobically in M9 mineral medium with glycerol and dimethyl sulfoxide as the energy substrates (7) was used for expression studies. For determining the K_D and V_max values for the various carboxylic acids, effectors (0–50 mM) were added to the M9/glycerol/Me₂SO growth medium from stock solutions (0.5 or 1 M, taken to pH 7 by NaOH) before growth. Samples of the cultures at an A_{578 nm} of 0.5–0.7 were withdrawn for measurement of -galactosidase activity (28).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
C₄-Dicarboxylates as Effectors of DcuS-dependent Regulation—Expression of dcuB depends under anaerobic conditions only on active DcuR and reflects the degree of DcuS stimulation by external stimuli (7, 8). The expression of dcuB during anaerobic growth of E. coli IMW237 [MC4100 (dcuB'-'lacZ)] in the presence of a high concentration of various C₄-dicarboxylates and related compounds was measured in vivo (Table II). Each of the C₄-dicarboxylates, such as fumarate, succinate, or maleate, caused a high level of expression of the dcuB'-'lacZ reporter gene fusion. With the C₃- and C₅-dicarboxylates, malonate and glutarate, and with citrate, the level of expression was much lower. Potential effectors in which one or two of the carboxylate groups were replaced by the anionic sulfonate group had no stimulatory effect on dcuB'-'lacZ expression, whereas 3-nitropropionate caused strong stimulation. The amides and the methyl esters of the carboxylates retained only a low or no stimulating effect. The monocarboxylates, butyrate, propionate, acetate, and formate, did not lead to any significant induction, even when supplied in combination, e.g. propionate plus formate or acetate plus formate.

The Amino Acid Residues of the Periplasmic Domain Involved in Fumarate Binding—The binding site of the C₄-dicarboxylates in DcuS is proposed to be located in the periplasmic domain (7–9, 12). An alignment of the amino acid sequence of the periplasmic domain of DcuS from E. coli with those from the citrate sensor CitA from Klebsiella (4, 5) and other DcuS and CitA proteins revealed conserved residues in the members of the CitA/DcuS family (Fig. 2, unshaded boxes). Most of the conserved amino acid residues are hydrophobic (Ala, Gly, Ile, Leu, Val) and might be structurally relevant. Charged residues are conserved in both types of proteins (Asp¹⁰², Arg¹⁰⁷, His¹¹⁰, and Arg¹⁴⁷, according to the numbering of E. coli DcuS).

In addition (Fig. 2, shaded boxes), amino acid residues Met¹⁰³, Phe¹²⁰, Phe¹⁴⁹, and Gln¹⁵⁹ are conserved in the DcuS-like proteins, whereas Gly¹⁴³, Lys¹⁴⁹, Val/Ile¹⁵⁹, and Ser¹⁶⁴ (according to the numbering of E. coli CitA) are found in CitA-like proteins. In particular, the change of the pair Phe¹⁴⁹-Gln¹⁵⁹ in DcuS representing two polar but uncharged amino acid residues to Phe¹⁴⁹-Val/Ile¹⁵⁹ in the CitA proteins representing basic and hydrophobic residues seems to be coupled. This change from a polar/polar pair in DcuS to a basic/hydrophobic pair in CitA is indicative for the distinction of DcuS and CitA proteins (Fig. 2).

Positively charged amino acid residues would be expected to be important for the binding of the anionic C₄-dicarboxylates. Therefore, conserved and other significant Arg, Lys, and His residues of the DcuS periplasmic domain were replaced using site-directed mutagenesis by Ala or Ile residues. The mutant DcuS protein was tested for function in E. coli with a deletion of the wild-type chromosomal dcuS gene and a dcuB'-'lacZ reporter gene fusion (Fig. 3). In the dcuS deletion strain, the expression of the dcuB'-'lacZ reporter gene fusion was very low as predicted (<6% of the strain carrying wild-type chromosomal dcuS), whereas in the presence of the plasmid-encoded wild-type dcuS gene, the dcuB'-'lacZ reporter gene fusion was expressed at high levels. Of the mutations with substituted basic amino acid residues, R107A, H110A, R147A, and R147I caused a nearly complete loss of stimulation of dcuB'-'lacZ expression similar to the level of the dcuS mutant. In contrast, mutations in which the other positively charged residues were replaced (K76A, R139I, and K158A) showed stimulation of dcuB'-'lacZ expression similar to the wild type.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Expression of dcuB'-'lacZ as a function of succinate (•), maleate (), 3-nitropropionate (), or citrate () in the medium. Bacteria were grown anaerobically in M9 medium supplemented with glycerol and dimethyl sulfoxide as growth substrates, plus the effectors succinate (•), maleate (), 3-nitropropionate (), or citrate () to an A578 nm of 0.5. -galactosidase activity were then measured.

The results obtained for double mutants were consistent with those of the single mutations (Fig. 3). A combination of a mutation that resulted in a decrease in stimulation (e.g. R147I or F149K) with a second mutation, which was silent on its own, led to the same decrease in expression as the single former mutation. The combination of two silent mutations (e.g. A164S/L167Y) also did not affect dcuB'-'lacZ expression (Fig. 3).

NMR Studies on the Binding of Ligands to the Periplasmic Domain of DcuS—The isolated DcuS-PD was used for studies of the binding of the effectors to the domain by NMR spectroscopy (17). After the addition of fumarate, sharpening of peaks was observed for some of the amino acid residues of the periplasmic domain in ¹⁵N-¹H HSQC spectra (Fig. 4A). The affected residues cluster in a defined region in the structure of the periplasmic domain of DcuS involving amino acid residues 107–168 (Fig. 5A). This region corresponds to the binding pocket of citrate in the periplasmic domain of CitA (6). There were no chemical shift changes observed during the titration.

When DcuS-PD was titrated with D-tartrate chemical shift changes was observed for a number of residues (Figs. 4B and 5B and Supplemental Fig. 1). Most of these residues belong to the same region that was affected by fumarate binding. Such chemical shift changes were only observed for D-tartrate but not for L-tartrate. There was a set of common residues that were affected both by the fumarate and tartrate titration (Ala¹¹³, Lys¹²¹, Asn¹³⁴, Ala¹⁴⁵, and Thr¹⁵⁰). Other residues were specifically affected by fumarate (Arg¹⁰⁷, Ile¹¹⁶, Ala¹²⁸, Ala¹³⁶, Ala¹⁴³, Gln¹⁴⁴, Arg¹⁴⁷, and Phe¹⁴⁹) or D-tartrate addition (Val⁸⁹, Lys⁹¹, Leu⁹⁶, Phe⁹⁷, Val¹⁰⁰, His¹¹⁰, Gln¹¹⁴, Gln¹¹⁸, Asp¹²⁴, and Gly¹⁴⁰). A comparison of the affected residues indicates that fumarate and tartrate bind to the same positively charged binding pocket in the DcuS-PD domain (Fig. 5C). In addition, some residues located outside this defined binding pocket are specifically affected by D-tartrate and more nonpolar residues are affected by fumarate than D-tartrate, the latter containing two additional hydroxyl groups.

¹⁵N-¹H HSQC spectra of mutant DcuS(F120M)-PD and DcuS(H110A)-PD showed a very small chemical shift dispersion characteristic for unfolded proteins (Supplemental Fig. 2A). On the other hand, for DcuS(R147A)-PD, a broadening of the NH resonances was observed (Supplemental Fig. 2B). This indicates a higher oligomerization state, in agreement with an increased rate of precipitation of DcuS(R147A)-PD.

Effect of the R147A Mutation on Autophosphorylation and Histidine Kinase Activity of DcuS—The mutant protein DcuS(R147A), which lost in vivo activity for stimulation of gene expression nearly completely, was tested in vitro for autokinase activity and its response to fumarate. DcuS(R147A) with an N-terminal His₆ tag was overproduced and purified as a homogenous preparation. After reconstitution (12), the proteoliposomes were incubated with [-³³P]ATP in the presence of fumarate and the kinetics of DcuS(R147A) and DcuS phosphorylation were determined (Fig. 6). Phosphorylation of DcuS after separation of the proteins by SDS-polyacrylamide gel electrophoresis was quantified using a PhosphorImager. Similar to wild-type DcuS, DcuS(R147A) was capable of autophosphorylation. Phosphorylation of both proteins showed saturation after 45 min, but the rates and degrees of phosphorylation of the two proteins differed. Phosphorylation of the mutant protein amounted to 50% phosphorylation of the wild-type DcuS protein. When the purified response regulator DcuR was added to the phosphorylated samples, >80% radioactivity from DcuS and from DcuS(R147A) was transferred to DcuR within 1 min, which indicated that DcuS(R147A) was not impaired in phosphoryl transfer to DcuR.

Lack of Stimulation of Histidine Kinase Activity of DcuS(R147A)—The stimulation of DcuS and DcuS(R147A) autophosphorylation by C₄-dicarboxylates was tested. The relative amounts of the phosphorylated proteins were determined after 30 min of incubation, which provides a good measure for the differences in phosphorylation (Fig. 7). In the absence of a stimulus, wild-type and mutant DcuS showed the same degree of phosphorylation. Phosphorylation of wild-type DcuS increased 2-fold in the presence of the C₄-dicarboxylates fumarate, succinate, and malate, whereas citrate and acetate had no effect. Phosphorylation of DcuS(R147A), however, was not stimulated by the C₄-dicarboxylates or by other effectors. Therefore, the basic (unstimulated) activity and function of the kinase domain in DcuS(R147A) apparently is not affected but the effector-induced stimulation of phosphorylation is lost.

View larger version (127K): [in this window] [in a new window]   FIG. 2. Comparison of the amino acid sequences of the periplasmic sensor domains of C -dicarboxylate or citrate sensory histidine kinases. Amino acid residues conserved in both types of proteins are boxed; amino acids conserved 4in only one of the types of are boxed and shaded. Amino acid residues of E. coli DcuS that were changed by site-directed mutagenesis are indicated by arrows. BS, Bacillus subtilis; BH, Bacillus halodurans; CG, Corynebacterium glutamicum; CP, Clostridium perfringens; EC, E. coli; KP, Klebsiella pneumoniae; OI, Oceanobacillus ihejensis; SC, Streptomyces coelicolor; SF, Shigella flexneri; SP, Streptococcus pyogenes; ST, Salmonella typhimurium; VC, Vibrio cholerae.

View larger version (20K): [in this window] [in a new window]   FIG. 3. Effect of mutations in the DcuS-PD on DcuS-dependent dcuB'-'lacZ expression. E. coli IMW260 (dcuS) was transformed with low-copy number plasmids carrying the indicated mutated alleles of dcuS. -Galactosidase activity of cultures at an A578 nm of 0.5 was measured under standard conditions. The activity of E. coli IMW260 (dcuS) carrying the plasmid-encoded wild-type dcuS allele [p(dcuS+)] is shown for comparison.

View larger version (25K): [in this window] [in a new window]   FIG. 4. Interaction of fumarate and D-tartrate with the periplasmic domain of DcuS as characterized by NMR spectroscopy. NMR signals of backbone amides constitute excellent probes of complex formation, providing maps of the interaction interfaces. Changes in peak heights between 1H-15N HSQC spectra of free DcuS-PD and DcuS-PD in the presence of fumarate (30-fold excess) (A) and changes in 15N chemical shifts between the 1H-15N HSQC spectra of free DcuS-PD and DcuS-PD in the presence of D-tartrate (50-fold excess) (B) were observed. The concentration of DcuS-PD was 1.2 mM.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

DcuS tolerates a vast spectrum of substituents at the C2 or C3 position. Thus, small polar substitutions (hydroxy, thiolate, or amino groups) are tolerated, whereas large apolar substitutions like methyl groups prevent binding and stimulation. Thus, methylfumarate shows significantly decreased stimulation and dimethyl succinate has lost the capacity for stimulation. Citrate, on the other hand, which can be regarded as a C₄-dicarboxylate with an acetyl group as the substituent, is more efficient with respect to induction and apparent K_D than the C₃- and C₅-dicarboxylates, malonate and glutarate.

The C₄-Dicarboxylate Binding Site of DcuS—The sequence Arg-X₂-His-X₉-Phe-X₂₆-Arg-X-Phe presumably represents the signature sequence for binding of C₄-dicarboxylates by DcuS. The residues are conserved in DcuS-like proteins and have been shown by mutagenesis to be essential for DcuS function in vivo. Other residues (Met¹⁰³ and Gln¹⁵⁹), which are also conserved specifically in DcuS-like proteins (but not in CitA-like proteins), were not required for function when tested in vivo after mutation to Ala or Arg and Ala or Val, respectively.

The periplasmic domain of DcuS shares many conserved residues with the citrate sensor CitA from Klebsiella, and the residues required for citrate binding have been identified by mutagenesis (4, 5). CitA binds citrate presumably as a divalent anion (citrate^2–) and requires residues Arg¹⁰⁹, His¹¹², Arg¹⁵⁰, and Lys¹⁵² for binding. The former three residues are equivalent to the (essential) residues Arg¹⁰⁷, His¹¹⁰, and Arg¹⁴⁷ of DcuS. Residue Lys¹⁵² of CitA is replaced by the (essential) Phe residue in DcuS (Phe¹⁴⁹), which cannot be replaced by a Lys residue. The essential Phe¹²⁰ of DcuS obviously has no counterpart in CitA. Thus, Phe¹²⁰ is specific for DcuS, whereas Arg^107/109, His^110/112, and Arg^147/150 are present in DcuS and CitA as well and Phe¹⁴⁹-Lys¹⁵² are homologous replacements. This set of residues obviously is responsible for the binding of the C₄-dicarboxylate and citrate^2– in DcuS and CitA.

High resolution structures of the fumarate and succinate binding sites of fumarate reductases and succinate dehydrogenases have been determined. In Wolinella succinogenes and E. coli fumarate reductase, one carboxylate of the C₄-dicarboxylate is ligated by an Arg and a His residue and the second carboxylate is ligated by a His residue (Arg⁴⁰⁴ and His³⁶⁹ and His²⁵⁷ of W. succinogenes FrdA) (29, 30). Fumarate binding by DcuS might be similar, suggesting the binding of the first carboxylate by one Arg and one His residue and of the second carboxylate by the second essential Arg residue together with one of the conserved Phe residue. Arg³⁰¹ from the active site of FrdA, which is required for fumarate reduction, has no homologue in DcuS.

Structural Properties of the C₄ Binding Site/Domain of DcuS and Comparison to the Citrate Binding Site of CitA—Structural information on the periplasmic domains of DcuS and of CitA, which has become available recently (6, 17), will help to understand C₄-dicarboxylate binding in future. The structure of the periplasmic domain of DcuS with bound fumarate, which is not available yet due to the low affinity binding of the C₄-dicarboxylates, will be required. However, from the comparison of the binding pockets of DcuS and CitA where the x-ray structure is available (6), it was tempting to propose a binding model of fumarate to DcuS. Indeed, the residues that are involved in the binding of one of the carboxylate groups of citrate (Lys¹⁵² Phe¹⁴⁹ and Ser¹⁶⁷ Ala¹⁶⁵) have lost their positive charge in DcuS so that one could speculate that the residues of the other two carboxylate groups are used by DcuS to bind the two carboxylate groups of fumarate. However, due to the fact that the nonphysiological molybdate is bound very close to the citrate in the CitA, we refrain from a detailed discussion of binding modes.

C₄-Dicarboxylate Binding Site and Signal Transfer in DcuS—Mutation in the fumarate binding site of DcuS affected only C₄-dicarboxylate-dependent expression in vivo and the fumarate-stimulated kinase activity in vitro. The basic kinase activity itself and the phosphoryl transfer to DcuR were not impaired, which demonstrated that kinase activity per se is independent of the sensory domain. In addition, the experiments directly show that binding of fumarate to the (periplasmic) binding domain controls the activity of the (cytoplasmic) kinase and transmitter domains. Future experiments will aim at an understanding of the reactions and structural changes induced by fumarate binding and their transmission from the binding site to the transmitter domain of DcuS.

View larger version (33K): [in this window] [in a new window]   FIG. 5. Structure of the periplasmic domain of DcuS showing the residues most affected in peak height by fumarate (A) or in chemical shift by D-tartrate addition (B). Residues highlighted in A are Arg107, Ala113, Ile116, Lys121, Ala128, Asn134, Ala136, Ala143, Gln144, Ala145, Arg147, Phe149, and Thr150, and residues in B are Val89, Lys91, Leu96, Phe97, Val100, His110, Ala113, Gln114, Gln118, Lys121, Asp124, Asn134, Gly140, Ala145, and Thr150. C, electrostatic surface potential of the periplasmic domain of DcuS with positive and negative potential colored blue and red, respectively. Figures were generated using the program MOLMOL (31).

View larger version (15K): [in this window] [in a new window]   FIG. 6. Autophosphorylation of reconstituted DcuS and DcuS(R147A) in the presence of fumarate. Kinetics of DcuS () and DcuS(R147A) () autophosphorylation by [-33P]ATP in proteoliposomes relative to autophosphorylation of DcuS after 45 min (100%) are shown. DcuS-proteoliposomes and DcuS(R147A)-proteoliposomes containing 20 mM fumarate in the buffer and within the proteoliposomes were mixed with 100 µM [-33P]ATP. After 46 min of autophosphorylation, a 4-fold excess of His6-DcuR was added. At the time points indicated, aliquots (2 µg of DcuS protein) were withdrawn, quenched with SDS sample buffer, subjected to SDS-polyacrylamide gel electrophoresis, and evaluated using a PhosphorImager.

View larger version (19K): [in this window] [in a new window]   FIG. 7. Stimulation of DcuS and DcuS(R147A) autophosphorylation by C4-dicarboxylates, citrate, or acetate after reconstitution in liposomes. Phosphorylation of DcuS (gray bars) and DcuS(R147A) (black bars) by [-33P]ATP in proteoliposomes was measured in the presence of different carboxylic acids. After 30 min of incubation, the radioactivity bound by DcuS or DcuS(R147A) was determined after SDS-polyacrylamide electrophoresis and phosphorimaging analysis (see the legend to Fig. 6). The highest value obtained (DcuS with succinate) was set to 100%.

	FOOTNOTES

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1 and 2.

^¶ Supported by Emmy Noether Grant ZW 71/1-4 of the Deutsche Forschungsgemeinschaft.

^|| To whom correspondence should be addressed: Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg-Universität Mainz, Becherweg 15, 55099 Mainz, Germany. Tel.: 49-6131-3923550; Fax: 49-6131-3922695; E-Mail: unden{at}uni-mainz.de.

¹ The abbreviations used are: DcuS, dicarboxylate uptake sensor (protein kinase); DcuS-PD, periplasmic domain of DcuS; DcuR, dicarboxylate uptake regulator response regulator); CitA, citrate sensor kinase; FNR, fumarate and nitrate reductase regulator; PAS domain, Per-Arnt-Sim domain; PYP, photoactive yellow protein.

² T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco.

	ACKNOWLEDGMENTS

 
We are grateful to R. Zentel (Mainz) for calculating distances of the carboxylate groups in the dicarboxylates. Karen A. Brune is acknowledged for critically reading the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Parkinson, J. S., and Kofoid, E. C. (1992) Annu. Rev. Genet. 26, 71–112[CrossRef][Medline] [Order article via Infotrieve]
2. Aizawa, S. I., Harwood, R. J., and Kadner, J. (2000) J. Bacteriol. 182, 1459–1471[Free Full Text]
3. Mizuno, T. (1997) DNA Res. 4, 161–168[Abstract]
4. Kaspar, S., Perozzo, R., Reinelt, S., Meyer, M., Pfister, L., Scapozza, L., and Bott, M. (1999) Mol. Microbiol. 33, 858–872[CrossRef][Medline] [Order article via Infotrieve]
5. Gerharz, T., Reinelt, S., Kaspar, S., Scapozza, L., and Bott, M. (2003) Biochemistry 42, 5917–5924[CrossRef][Medline] [Order article via Infotrieve]
6. Reinelt, S., Hofmann, E., Gerharz, T., Bott, M., and Madden, D. R. (2003) J. Biol. Chem. 278, 39189–39196[Abstract/Free Full Text]
7. Zientz, E., Bongaerts, J., and Unden, G. (1998) J. Bacteriol. 180, 5421–5425[Abstract/Free Full Text]
8. Golby, P., Davies, S., Kelly, J. R., Guest, J. R., and Andrews, S. C. (1999) J. Bacteriol. 181, 1238–1248[Abstract/Free Full Text]
9. Janausch, I. G., Zientz, E., Tran, Q. H., Kröger, A., and Unden, G. (2002) Biochim. Biophys. Acta 1553, 39–56[Medline] [Order article via Infotrieve]
10. Engel, P., Krämer, R., and Unden, G. (1994) Eur. J. Biochem. 222, 605–614[Medline] [Order article via Infotrieve]
11. Six, S., Andrews, S. C., Unden, G., and Guest, J. R. (1994) J. Bacteriol. 176, 6470–6478[Abstract/Free Full Text]
12. Janausch, I. G., Garcia-Moreno, I., and Unden, G. (2002b) J. Biol. Chem. 277, 39809–39814[Abstract/Free Full Text]
13. Abo-Amer, E. E., Munn, J., Jackson, K., Aktas, M., Golby, P., Kelly., D. J., and Andrews, S. C. (2004) J. Bacteriol. 186, 1879–1889[Abstract/Free Full Text]
14. Janausch, I. G., Garcia-Moreno, I., Zeuner, Y., Lehnen, D., and Unden G. (2004) Microbiology 150, 877–883[Abstract/Free Full Text]
15. Bott, M., Meyer, M., and Dimroth, P. (1995) Mol. Microbiol. 18, 533–546[CrossRef][Medline] [Order article via Infotrieve]
16. Parac, T. N., Coligaev, B., Zientz, E., Unden, G., Peti, W., and Griesinger, C. (2001) J. Biomol. NMR 19, 91–92[CrossRef][Medline] [Order article via Infotrieve]
17. Pappalardo, L., Janausch, I. G., Vijayan, V., Zientz, E., Junker, J., Peti, W., Zweckstetter, M., Unden, G., and Griesinger, C. (2003) J. Biol. Chem. 178, 39185–39188
18. Borgstahl, G. E. O., Williams, D. R., and Getzoff, E. D. (1995) Biochemistry 34, 6278–6287[CrossRef][Medline] [Order article via Infotrieve]
19. Kaspar, S., and Bott, M. (2002) Arch. Microbiol. 177, 313–321[CrossRef][Medline] [Order article via Infotrieve]
20. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
21. Chen, W., and Kuo, T. (1993) Nucleic Acids Res. 21, 2260[Free Full Text]
22. Hanahan, D. (1983) J. Mol. Biol. 166, 557–565[Medline] [Order article via Infotrieve]
23. Inoue, H., Nojima, H., and Okayama, H. (1990) Gene (Amst.) 96, 23–28[CrossRef][Medline] [Order article via Infotrieve]
24. Tran, Q. H., and Unden, G. (1998) Eur. J. Biochem. 251, 538–543[Medline] [Order article via Infotrieve]
25. Holloway, P. W. (1973) Anal. Biochem. 53, 304–308[CrossRef][Medline] [Order article via Infotrieve]
26. Rigaud, J. L., Pitard, B., and Levy, D. (1995) Biochim. Biophys. Acta 1231, 223–246[Medline] [Order article via Infotrieve]
27. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) J. Biomolecular NMR. 6, 277–293
28. Miller, J. H. (1992) A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
29. Lancaster, C. R. D., Gro, R., and Simon, J. (2001) Eur. J. Biochem. 268, 1820–1827[Medline] [Order article via Infotrieve]
30. Iverson, T. M., Luna-Chavez, C., Cecchini, G., and Rees, D. C. (1999) Science 284, 1961–1966[Abstract/Free Full Text]
31. Koradi, R., Billeter, M., and Wüthrich, K. (1996) J. Mol. Graph. 14, 29–32

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. Kleefeld, B. Ackermann, J. Bauer, J. Kramer, and G. Unden The Fumarate/Succinate Antiporter DcuB of Escherichia coli Is a Bifunctional Protein with Sites for Regulation of DcuS-dependent Gene Expression J. Biol. Chem., January 2, 2009; 284(1): 265 - 275. [Abstract] [Full Text] [PDF]

				P. Scheu, S. Sdorra, Y.-F. Liao, M. Wegner, T. Basche, G. Unden, and W. Erker Polar accumulation of the metabolic sensory histidine kinases DcuS and CitA in Escherichia coli Microbiology, August 1, 2008; 154(8): 2463 - 2472. [Abstract] [Full Text] [PDF]

				N. Moker, P. Reihlen, R. Kramer, and S. Morbach Osmosensing Properties of the Histidine Protein Kinase MtrB from Corynebacterium glutamicum J. Biol. Chem., September 21, 2007; 282(38): 27666 - 27677. [Abstract] [Full Text] [PDF]

				J. Kramer, J. D. Fischer, E. Zientz, V. Vijayan, C. Griesinger, A. Lupas, and G. Unden Citrate Sensing by the C4-Dicarboxylate/Citrate Sensor Kinase DcuS of Escherichia coli: Binding Site and Conversion of DcuS to a C4-Dicarboxylate- or Citrate-Specific Sensor J. Bacteriol., June 1, 2007; 189(11): 4290 - 4298. [Abstract] [Full Text] [PDF]

				N. Moker, J. Kramer, G. Unden, R. Kramer, and S. Morbach In Vitro Analysis of the Two-Component System MtrB-MtrA from Corynebacterium glutamicum J. Bacteriol., May 1, 2007; 189(9): 3645 - 3649. [Abstract] [Full Text] [PDF]

				O. B. Kim and G. Unden The L-Tartrate/Succinate Antiporter TtdT (YgjE) of L-Tartrate Fermentation in Escherichia coli J. Bacteriol., March 1, 2007; 189(5): 1597 - 1603. [Abstract] [Full Text] [PDF]

				C. Baraquet, L. Theraulaz, M. Guiral, D. Lafitte, V. Mejean, and C. Jourlin-Castelli TorT, a Member of a New Periplasmic Binding Protein Family, Triggers Induction of the Tor Respiratory System upon Trimethylamine N-Oxide Electron-acceptor Binding in Escherichia coli J. Biol. Chem., December 15, 2006; 281(50): 38189 - 38199. [Abstract] [Full Text] [PDF]

				T. Mascher, J. D. Helmann, and G. Unden Stimulus Perception in Bacterial Signal-Transducing Histidine Kinases Microbiol. Mol. Biol. Rev., December 1, 2006; 70(4): 910 - 938. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 280/21/20596    most recent M502015200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kneuper, H. Articles by Unden, G. Search for Related Content PubMed PubMed Citation Articles by Kneuper, H. Articles by Unden, G. Social Bookmarking                      What's this?