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Originally published In Press as doi:10.1074/jbc.M605854200 on November 3, 2006

J. Biol. Chem., Vol. 282, Issue 2, 1175-1182, January 12, 2007
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Residues His-15 and Arg-17 of HPr Participate Differently in Catabolite Signal Processing via CcpA*

Nicola Horstmann1, Gerald Seidel1, Lwin-Mar Aung-Hilbrich, and Wolfgang Hillen2

From the Lehrstuhl für Mikrobiologie, Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany

Received for publication, June 19, 2006 , and in revised form, October 2, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The carbon catabolite control protein A (CcpA) senses the physiological state of the cell by binding several effectors and responds with differential regulation of many genes in Bacilli. HPr-Ser46-P or Crh-Ser46-P interact with CcpA and stimulate binding to catabolite responsive elements. In addition, the glycolytic intermediates fructose 1,6-bisphosphate (FBP) and glucose 6-phosphate (Glc-6-P) stimulate HPr-Ser46-P but not Crh-Ser46-P binding to CcpA. The mechanisms by which coeffector binding to CcpA is linked to differential gene expression are unclear. To address this question we mutated residues participating in the interaction between HPr-Ser46-P or Crh-Ser46-P and CcpA and analyzed their effects on CcpA binding and stimulation of cre binding by surface plasmon resonance. The HPrH15A and CcpAD297A mutations do not affect complex formation but abolish FBP and Glc-6-P stimulation. Likewise, the CrhQ15H mutant becomes sensitive to these glycolytic intermediates. Hence, the contact of HPrHis-15 to Asp-297 in CcpA is a determinant for HPr specific FBP and Glc-6-P stimulation. The HPrR17A and -K mutants are both strongly impaired in stimulation of CcpA binding to cre, but only HPrR17A is defect in binding to CcpA indicating that these residues affect allostery of CcpA. Mutations of the residues of CcpA, which contact Arg-17 of HPr, exhibit differential effects on regulation of catabolic genes. Taken together, His-15 of HPr processes sensing information, while Arg-17 is involved in determining the genetic output.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacteria efficiently regulate gene expression to adjust to different environmental conditions. Carbon catabolite regulation (CCR)3 represents a main global regulatory mechanism enabling bacteria to exclusively utilize the carbon source providing the fastest growth rate from a mixture of supplies (1, 2). CCR in Gram-positive bacteria with low GC content is mediated by the global transcriptional regulator catabolite control protein A (CcpA) (3, 4). It receives signals representing the energy state of the cell and the availability of carbon sources and responds by binding to catabolite response elements (cre), thereby activating or repressing more than 300 genes in Bacillus subtilis (5, 6). CcpA is a member of the LacI/GalR familiy of transcriptional regulators (7). In contrast to other family members CcpA predominantly requires the phosphoproteins HPr-Ser46-P or Crh-Ser46-P as coeffectors, but small ligands like fructose 1,6-bisphosphate (FBP) or glucose 6-phosphate (Glc-6-P) can also stimulate CcpA activity (8-11).

HPr plays a dual role in carbon utilization. The His-phosphorylated form of HPr, HPr-His15-P, is a component of the phosphoenolpyruvat:sugar phosphotransferase system (PTS) for sugar uptake and can also phosphorylate regulatory proteins (1). When HPr is phosphorylated at Ser-46 by FBP stimulated HPr kinase/phosphorylase (12) it assumes merely regulatory functions as a cofactor for CcpA. Crh is homologous to HPr, but it contains Gln instead of His at position 15 and, hence, is not active in the PTS. It can only be phosphorylated at Ser-46 catalyzed by HPr kinase/phosphorylase leading to stimulation of CcpA binding to cre (9).

The roles of these proteins for regulation are not fully understood, but their contributions to CCR in B. subtilis are clearly different: Crh-Ser46-P cannot completely substitute for HPr-Ser46-P in the regulation of many promoters, while only citM is known to depend exclusively on Crh-Ser46-P (9, 13, 14). The expression levels of Crh and HPr are quite different. The presence of glucose or other PTS substrates leads to an about 100-fold excess of HPr over Crh (15). Moreover, Crh-Ser46-P binds CcpA with an about 4-fold lower affinity than HPr-Ser46-P (11). This is in accordance with the crystal structures of the corresponding ternary complexes, in which the residues Gln-15, Arg-17, and Ala-20 of Crh-Ser46-P form weaker or no contacts to CcpA as compared with the respective residues His-15, Arg-17, and Thr-20 of HPr-Ser46-P (16, 17). Furthermore, the affinity of CcpA to HPr-Ser46-P, but not to Crh-Ser46-P, is increased by FBP or Glc-6-P, reducing the amount of HPr-Ser46-P necessary for cre binding about 10-fold (11).

The functions of the HPr residues His-15 and Arg-17 in the PTS have been analyzed in several studies (18-20). His-15 is the site of phosphotransfer and Arg-17 is important for binding to EI and EII (19, 20). A role of the His-15 residue in CCR has been demonstrated, while HPrR17 mutants were not yet tested for their influence on CCR. We describe here the molecular mechanisms by which both residues contribute to regulation of carbon metabolism.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction—Strains and plasmids used in this study are listed in Table 1. The ccpA mutants were obtained from two-step mutagenesis, restricted with XbaI and ClaI and cloned into pWH143, a pHT304 derivative, to complement a B. subtilis {Delta}ccpA mutant strain in vivo. For overproduction, his-tagged ccpA mutants were cloned into pWH653 using XbaI and NotI. The mutagenesis of ptsH from Bacillus megaterium and B. subtilis and crh from B. subtilis was performed by two-step mutagenesis. The resulting PCR-fragments were restricted with NdeI and BamHI and cloned into pET3c.


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  		TABLE 1
Plasmids and strains used in this study

 
beta-Galactosidase Activity Measurements—Cells were grown in LB or in LB with 1% glucose (ackA'::lacZ and alsS'::lacZ). The beta-galactosidase activity expressed by xynP::lacZ was measured in CSK minimal medium (C minimal medium supplemented with potassium succinate and potassium glutamate), in CSK medium with 0.2% xylose or in CSK medium with 0.2% xylose and 1% glucose. All cultures were initially inoculated to A600 = 0.1 and grown at 37 °C to the exponential growth phase (xynP'::lacZ) or to the stationary phase (alsS'::lacZ and ackA'::lacZ). beta-Galactosidase acivitities were determined as described (21). The resulting beta-galactosidase activities in Miller units were normalized to the fully induced or fully activated states (100% value) of the strain with His-tagged wild type ccpA. Full induction for xynP is obtained in CSK medium supplemented with 0.2% (w/v) xylose. For alsS and ackA full activation is achieved in LB medium supplemented in 1% (w/v) glucose. The data represent average values of three different clones, respectively, with standard deviation and are confirmed by at least two independent measurements.

Protein Preparation—C-terminally his-tagged CcpA from B. megaterium was expressed in B. megaterium WH419/pWH1499. C-terminally his-tagged CcpA from B. subtilis was expressed in B. megaterium WH419/pWH1543, while the CcpA mutant was expressed in E. coli FT1/pWH653-D297A (Table 1). Expression was induced at an A600 between 0.4 and 0.6 with 0.5% (w/v) xylose (pWH1543) or with 1 mM isopropyl beta-D-thiogalactopyranoside(pWH653). Both proteins were purified as described previously (11).

HPr from B. megaterium and B. subtilis and Crh from B. subtilis were expressed in Escherichia coli FT1/pWH1576, FT1/pWH466, and FT1/467, respectively, after induction at A600 = 0.4–0.6 with 1 mM isopropyl beta-D-thiogalactopyrano-side. All proteins are highly expressed upon induction and did not form inclusion bodies. The crude extract was incubated on ice with 5 µg/ml RNaseA and 10 µg/ml DNaseI (Sigma, Munich, Germany) and subsequently centrifuged to remove cell debris. A prepurification was achieved by heat denaturation for 20 min at 70 °C and 65 °C respectively, except for the Crh mutant Q15H, which emerged as instable at high temperatures. After centrifugation from the precipitated cellular proteins, phosphorylation of either protein was performed in the crude lysate with Crh-Q15H and in the prepurified crude lysates with all other HPr and Crh proteins using HPr kinase extract as described (22). Further purification was obtained by anion exchange chromatography on Sepharose QFF (GE Healthcare, Freiburg, Germany) and subsequent gel permeation chromatography on Superdex G75 (GE Healthcare). The proteins are fully phosphorylated and purified to homogeneity. The activity was assumed to be 100%. Protein concentrations were measured using a Bio-Rad (Munich, Germany) Bradford dye binding assay with bovine serum albumin as standard.

Surface Plasmon Resonance (SPR) Measurements—SPR measurements were performed on a BIAcoreX instrument operated at 25 °C (BIAcoreX, Uppsala, Sweden) as described in Ref. 11 with one exception. Initially, cre DNA was coupled on a CM5 chip using CTAB micelles, but this method lead to varying amounts of active DNA on the chip. Therefore, about 3000 RU of neutravidin, a streptavidin analog that reduces nonspecific interactions, was coupled on a CM5 chip activated with N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminoproply)carbodiimide. Subsequently, 75 RU of 5'-biotinylated xylAcre or unspecific DNA, respectively, was captured. The depicted overlays are representative traces. All quantitative and qualitative measurements were done at least twice and yielded reproducible data. The equilibrium constants and the mean root square deviations in Table 2 were derived from at least two titrations.


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  		TABLE 2
Equilibrium constants of CcpA complexes with HPr and Crh variants from B. megaterium and B. subtilis and of the CcpAD297A mutant with HPr-Ser46-P and Crh-Ser46-P

 
CD Measurements—The protein solutions were dialyzed from Biacore running buffer (11) in 10 mM sodium phosphate buffer with 150 mM NaF at pH 7.4 prior to the measurements. CD spectra from 20 µM wild type or mutant HPr-Ser46-P were recorded on a Jasco J-715 spectropolarimeter at 25 °C (Jasco, Gross-Umstadt, Germany). The spectra were processed with the program J-700 for Windows Standard Analysis version 1.50.01. The depicted overlays are representative traces. The spectra were recorded twice and yielded reproducible data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
HPr-Ser46-P and Crh-Ser46-P Mutants with Changes in Residues Arg-17 or His-15 Show Different Affinities for CcpA—We introduced several different mutations to determine the contributions of the residues at positions 15 and 17 of HPr and Crh to CcpA binding: 1) residue Arg-17 was changed to alanine and lysine in B. megaterium HPr to investigate the impact of charge and length of the side chain, 2) Residue His-15 in B. megaterium HPr was mutated to alanine and glutamate to determine the contribution of the imidazole moiety and mimic the presence of phosphate, 3) His-15 in B. subtilis HPr was substituted by the corresponding residue in Crh (glutamine) and vice versa to determine their contributions to the activity differences of these effectors. The wild type and mutant proteins were over-expressed in E. coli, phosphorylated at Ser-46, and both forms were purified to homogeneity. Native polyacrylamide gel electrophoresis revealed that the HPr-Ser46-P and Crh-Ser46-P preparations did not contain any visible amounts of the respective non-phosphorylated form (data not shown). Their interaction with CcpA from B. megaterium or B. subtilis was analyzed by SPR using the corresponding CcpA coupled to CM5 chips. Binding of the phosphorylated HPr or Crh variants was initially examined at concentrations of 15 µM (Fig. 1). Non-phosphorylated HPr or Crh variants did not interact with CcpA even at a concentration of 100 µM (data not shown). The SPR signals obtained with the HPrH15Q-Ser46-P mutant and the CrhQ15H-Ser46-P mutant from B. subtilis showed similar intensities like the respective wild type proteins. Therefore, the binding constants of these mutants to CcpA were quantified (Fig. 2) and are presented in Table 2. Neither the exchange of histidine at position 15 in HPr-Ser46-P to glutamine nor the complementary exchange of glutamine 15 to histidine in Crh-Ser46-P affected the interaction with CcpA. The respective KD values are similar to those obtained with the wild type proteins. Likewise, the mutation of HPrH15 from B. megaterium to ala-nine had only a negligible influence on its affinity for CcpA. The introduction of a negative charge in HPrH15E-Ser46-P from B. megaterium, however, impaired CcpA binding drastically, so that no saturation was obtained in the titration. Mutation of HPrR17 from B. megaterium to alanine also decreased CcpA binding of the phosphorylated form. In contrast, the conservative mutation in HPrR17K-Ser46-P affected CcpA interaction only slightly. Titrations (Fig. 2) of CcpA with this mutated phosphoprotein revealed a 2.6-fold decrease in affinity compared with the wild type (Table 2). We conclude that mutation of HPr at residue 15 to a neutral amino acid does not affect the affinity of the phosphoprotein to CcpA, while the introduction of a negative charge impairs CcpA binding. The conservative replacement of HPr residue Arg-17 in HPrR17K-Ser46-P shows no influence on the interaction with CcpA.


Figure 1
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  		FIGURE 1.
Qualitative analyses of effector binding to CcpA. Sensorgrams in the left diagram were obtained from the SPR analyses of 100 µM HPr and 15 µM HPr-Ser46-P, HPrH15A-Ser46-P, HPrR17K-Ser46-P, HPrH15E-Ser46-P, or HPrR17A-Ser46-P, respectively, from B. megaterium on a chip with immobilized CcpA(His6) from B. megaterium. The diagram on the right side shows sensorgrams from 100µM HPr and 15µM HPr-Ser46-P, Crh-Ser46-P, HPrH15QSer46-P, or CrhQ15H-Ser46-P, respectively, from B. subtilis analyzed on a chip with immobilized CcpA(His)6 from B. subtilis. The corresponding effector protein is indicated above each sensorgram.

 


Figure 2
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  		FIGURE 2.
Determination of KD values for CcpA: effector interactions. Left diagram, Immobilized CcpA(His)6 from B. megaterium was titrated with HPr-Ser46-P, HPrR17K-Ser46-P, and HPrH15A-Ser46-P from B. megaterium. Right diagram, CcpA(His)6 from B. subtilis was titrated with HPr-Ser46-P, Crh-Ser46-P, HPrH15Q-Ser46-P, and CrhQ15H-Ser46-P from B. subtilis. SPR response units were plotted versus the coeffector concentrations and fitted to the Langmuir equation for a 1:1 binding reaction per CcpA subunit.

 


Figure 3
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  		FIGURE 3.
CD spectra of wild type and mutant HPr-Ser-46-P. The CD spectra were recorded from 20 µM samples of wildtype HPr-Ser46-P (solid line), HPrH15E-Ser46-P (dashed line), and HPrR17A-Ser46-P (dotted line).

 
HPrH15E-Ser46-P and HPrR17A-Ser46-P Are Correctly Folded—To exclude that the inactivity of the mutants HPrH15E-Ser46-P and HPrR17A-Ser46-P in the in vitro experiments results from improper folding we recorded CD spectra from wild type HPr-Ser46-P and the respective mutant phospho-proteins at concentrations of 20 µM (Fig. 3). The spectra are identical with each other and the ones of E. coli and Streptomyces coelicolor HPr (23, 24). We therefore assume that all proteins are correctly folded.

Effects of His-15 and Arg-17 Mutations in HPr and Crh on Stimulation of CcpA Binding to cre—HPr-Ser46-P and Crh-Ser46-P stimulate binding of CcpA to cre from xylA by formation of the respective ternary complexes. To elucidate the effects of the HPr or Crh mutations on the formation of these complexes, we performed a qualitative SPR analysis with xylA-cre immobilized on CM5 chips. Fig. 4 shows the stimulation of DNA binding by 15 µM of the HPr-Ser46-P variants. In good agreement with their affinities for CcpA, HPrH15A-Ser46-P, HPrH15Q-Ser46-P, and CrhQ15H-Ser46-P stimulated cre binding as well as the respective wild types. Moreover, the HPrH15E-Ser46-P and HPrR17A-Ser46-P mutants did not stimulate xylAcre binding in accordance with their reduced affinity for CcpA. In contrast, the HPrR17K-Ser46-P variant stimulated cre binding only poorly, although it bound CcpA well (see Figs. 2 and 4).

The CcpAD297A Mutation Does Not Affect the Affinity to HPr-Ser46-P or Crh-Ser46-P—Since residue Asp-297 in CcpA interacts directly with His-15 of HPr-Ser46-P (16) we mutated Asp-297 to alanine and analyzed the influence of this residue on the affinity of HPr-Ser46-P and Crh-Ser46-P for CcpA. The CcpA mutant protein was overexpressed in E. coli, purified, and coupled to a CM5 chip for SPR analysis. Like wild type CcpA, CcpAD297A did not interact with non-phosphorylated HPr and Crh (data not shown). CcpAD297A exhibited a similar affinity for both effectors as wild type CcpA (11) (Table 2), and HPr-Ser46-P stimulated CcpAD297A binding to xylAcre as efficiently as the wild type (data not shown).


Figure 4
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  		FIGURE 4.
Surface plasmon resonance analyses of stimulatory effects of HPr mutants on CcpA-cre interaction. Sensorgrams were obtained from the interaction analysis of cre with complexes formed from CcpA(His6) (10 nM)in the presence of 15 µM of HPr, HPr-Ser46-P, or different HPr mutants from B. megaterium as depicted next to each sensorgram.

 
Residues His-15 in HPr and Asp-297 in CcpA Are Essential for FBP and Glc-6-P Stimulation—We have recently identified FBP and Glc-6-P as specific effectors only for HPr-Ser46-PCcpA but not for Crh-Ser46-P-CcpA binding. Since HPr and Crh have a high sequence identity, we analyzed the effects of FBP and Glc-6-P on the interactions of the phosphorylated HPr and Crh mutants with CcpA by SPR. We also studied the interactions of HPr-Ser46-P and Crh-Ser46-P with the CcpAD297A mutant. The running buffers and the analyte mixtures contained FBP or Glc-6-P to avoid dissociation during the measurements. The concentrations of the phosphoproteins were adjusted to yield about 20% occupation of CcpA (40–50 RU) in the absence of sugar phosphates. Fig. 5 shows sensorgrams resulting from injections of 2 µM HPr-Ser46-P or 3 µM HPrH15A-Ser46-P from B. megaterium and 1 µM HPr-Ser46-P, 1 µM HPrH15Q-Ser46-P, 10 µM Crh-Ser46-P, or 8 µM CrhQ15H-Ser46-P from B. subtilis titrated with 5–40 mM of FBP. FBP and Glc-6-P increased HPr-Ser46-P and HPrR17KSer46-P (data not shown) binding to CcpA, but no influence was found for HPrH15A-Ser46-P, HPrH15Q-Ser46-P, and Crh-Ser46-P. Introduction of the histidine residue at position 15 of Crh in the CrhQ15H-Ser46-P mutant led to stimulation of CcpA binding by FBP and Glc-6-P. The KD value for CcpACrhQ15H-Ser46-P interaction decreased about 3-fold to 10.3 µM indicating a higher affinity in the presence of 40 mM FBP (Table 2). The sensorgrams for HPr-Ser46-P and HPrH15ASer46-P (Fig. 6) and HPrH15Q-Ser46-P and CrhQ15H-Ser46-P (data not shown) indicate that mutation of His-15 in HPr abolished FBP and Glc-6-P stimulation of cre binding. These results demonstrate that the His-15 residue in HPr mediates FBP stimulation, and its absence in Crh causes the lack of FBP stimulation.

We presumed that the CcpAD297-HPrH15 contact mediates the FBP and Glc-6-P effect in the CcpA-HPr-Ser46-P complex. Therefore, we analyzed the CcpAD297A mutant for stimulation by these compounds. FBP and Glc-6-P did not enhance HPr-Ser46-P binding to CcpAD297A (see Fig. 7) or HPr-Ser46-P-CcpAD297A-cre complex formation (data not shown). Thus, the CcpA residue Asp-297 is necessary for the stimulation by FBP and Glc-6-P of CcpA-HPr-Ser46-P binding to cre.


Figure 5
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  		FIGURE 5.
Effects of FBP and Glc-6-P on binding of HPr-Ser46-P and Crh-Ser46-P mutants to CcpA. A–C, sensorgrams were obtained from SPR analyses of the interaction of CcpA(His)6 with HPr-Ser46-P (2 µM) and HPrH15ASer46-P (3 µM) from B. megaterium (A), with HPr-Ser46-P (1 µM) and Crh-Ser46-P (10 µM) from B. subtilis (B), and with HPrH15Q-Ser46-P (1 µM) and CrhQ15H-Ser46-P (8 µM) from B. subtilis in the presence of increasing FBP concentrations as indicated (C). The concentrations of the phosphoproteins were adjusted to yield about 20% occupation of CcpA (40–50 RU) in the absence of sugar phosphates.

 
Mutational Analysis of CcpA Residues Contacting Arginine 17 of HPr-Ser46-P—The crystal structure of the CcpA-HPr-Ser46-P-cre complex from B. megaterium shows interactions of Arg-17 in HPr-Ser46-P with residue Asp-84 of one CcpA core subdomain, and the residues Asp-69' and Asp-99' from the other CcpA subdomain. The two N-terminal core subdomains of CcpA are supported in their cre binding conformations by these contacts (16). To clarify the importance of these interactions for CCR we altered Asp-70, Asp-85, and Asp-100 in B. subtilis CcpA to positively charged arginine residues. The generated B. subtilis strains express the single mutants CcpAD70R, -D85R, and -D100R, the double mutants CcpAD70R-D100R and -D85R-D100R, and the triple mutant CcpAD70R-D85RD100R. Western blotting confirmed that all CcpA variants were expressed at the same level as wild type CcpA (data not shown). We examined the effects of these mutations on three different CCR controlled promoters: xynP is repressed by CcpA; ackA is directly activated by CcpA; and alsS is indirectly activated by CcpA (13, 25-27). The results are listed in Table 3 and indicate quite different effects of these mutations on transcription of these three promoters. CcpA shows a small activation of ackA in the absence of glucose, which is not seen in the {Delta}ccpA strain. The same effect can be seen for the xynP fusion when induced with xylose (compare ccpA wt and {Delta}ccpA in Table 3) but not for the alsS fusion. In detail, the CcpA point mutants showed different extents of reduced repression of xynP. CcpAD100R regulated xynP most efficiently but also exhibited a somewhat decreased expression of xynP in the absence of glucose. This indicates an increased contribution from glucose-independent repression for this mutant. CcpAD70R showed clearly reduced and CcpAD85R almost no repression of xynP. CcpAD85R did not activate alsS expression and CcpAD70R was less active than CcpAD100R. Unlike xynP, however, the latter showed no glucose independent effect. In contrast, these CcpA mutants activated ackA as well as wild type CcpA. The double mutants were inactive for xynP repression, fully active for ackA activation, and partially active for alsS activation. The triple mutant did not regulate any of the three promoters investigated here. In conclusion, the residues Asp-70, Asp-85, and Asp-100 in CcpA are clearly of different importance for the promoters studied here, which may indicate different mechanisms of regulation by CcpA.


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  		TABLE 3
Normalized beta-galactosidase activities of B. subtilis wild type (wt) or ccpA mutant strains carrying xynP', ackA', or alsS'::lacZ fusions LB, Luria Bertani broth; glc, 1% glucose; xyl, 0.5% xylose.

 


Figure 6
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  		FIGURE 6.
SPR analyses of HPr-Ser46-P- and HPrH15A-Ser46-P-CcpA-cre complex formation in the presence of FBP. The analyses were conducted on a CM5 chip with immobilized xylAcre. The sensorgrams in the left diagram were obtained from injections of 10 nM CcpA(His)6 (base line), 10 nM CcpA-(His)6 with 10 mM FBP (base line), and mixtures of 10 nM CcpA(His)6 and 5 µM HPr-Ser46-P from B. megaterium and increasing FBP concentrations. The right diagram shows the titration of xylAcre with 10 nM CcpAHis6 and 5 µM HPrH15A-Ser46-P and increasing FBP concentrations in the analyte solutions. The FBP concentrations are listed on the right side of each sensorgram.

 


Figure 7
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  		FIGURE 7.
Effects of FBP on HPr-Ser46-P and Crh-Ser46-P binding to CcpAD297A. Sensorgrams in the left diagram were obtained from SPR analyses of the interaction of immobilized CcpAD297A(His)6 with HPr-Ser46-P (1µM) in the presence of increasing FBP concentrations, and the right diagram shows the titration of CcpAD297A(His)6 with FBP in the presence of Crh-Ser46-P (4 µM). The corresponding FBP concentrations are depicted on the right side in the diagram.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
HPr residue Arg-17 is important for EI and EII recognition (19, 20) in the PTS phosphotransfer chain. We demonstrate here that it is also essential for binding of HPr-Ser46-P to CcpA and subsequent cre binding. The activities of different HPr mutants at position 17 clearly reveal two distinct activities, which depend on the interaction of that residue with CcpA: HPrR17A-Ser46-P binds only weakly to CcpA and does not stimulate cre binding. On the other hand, HPrR17K-Ser46-P binds only slightly less well than the wild type to CcpA, but it shows almost complete lack of stimulation of cre binding. Thus, the mere binding of CcpA is not sufficient for regulation. It must be accompanied by triggering a conformational change of CcpA to accomplish complex formation with cre. This property can be understood on the basis of the structures of the apo and DNA bound forms of CcpA. Like in other LacI/GalR family proteins, the N-terminal core subdomains of CcpA rotate toward each other to form the DNA binding conformation. In the previously studied members of this family (28, 29) this transition is triggered by binding of small effectors. Subdomain rotation in CcpA results from binding of the phospho-protein effectors HPr-Ser46-P or Crh-Ser46-P. Arg-17 may (Figs. 8 and 9) either trigger the rotation of subdomains or stabilize the DNA bound conformation via contacts to CcpA residues Asp-84, Asp-69', and Asp-99' (corresponding to Asp-85, Asp-70', and Asp-100' in B. subtilis CcpA) (16). In contrast, Arg-17 of Crh-Ser46-P interacts only with Asp-99' of CcpA (Asp-100' in B. subtilis CcpA) in the respective crystal structure (17). These contacts are only possible in the DNA binding conformation of two N-terminal core subdomains of CcpA. Visualization of the exchange R17K in the complex structure with HPrR17K-Ser46-P using the SwissPDB viewer program (30) suggests that Lys-17 can only contact Asp-84 of one N-terminal CcpA subdomain and hence cannot support the DNA binding structure. We conclude that bridging of the CcpA dimers by Arg-17 of the bound phospho-protein is necessary for binding to cre.

The triple mutant CcpAD70R-D85R-D100R does not regulate alsS, ackA, and xynP in vivo. The single mutants CcpAD70R, -D85R, or -D100R show strongly decreased regulation of xynP and alsS but full activation of ackA. This may hint toward the involvement of as of yet unknown mechanisms contributing to ackA activation, like contacts to RNA polymerase or other cofactors. This idea is supported by the observation that ackAcre2 and the upstream sequence are essential for ackA activation (31). HPr-Ser46-P or Crh-Ser46-P bound to CcpA and cre are arranged parallel to the DNA (see Fig. 8A), which would be a prerequisite for formation of a complex with RNAP or with a protein binding potentially upstream of ackAcre2.If this assumption is true, the affinity of CcpA for DNA may be less important for ackA activation than for repression of xynP or indirect activation of alsS.

Two modes of signal sensing are revealed by the mutations of His-15 in HPr-Ser46-P. Mutations to the neutral amino acids alanine or glutamine did not affect CcpA binding or interaction with cre (32), whereas mutation to the negatively charged glutamate residue strongly decreased CcpA and cre binding. The glutamate residue may resemble the His-15 phosphorylated form of HPr (32) indicating that HPr-Ser46-P only functions as an effector for CcpA when His-15 is not phosphorylated. On the other hand, His-15 exerts its regulatory effect via stimulation of CcpA and cre binding by FBP and Glc-6-P. These results explain the observations of previous Ni-NTA elution experiments (32), which indicated that HPrH15A-Ser46-P, unlike the wild type phospho-protein, is not retarded by CcpA in the presence of FBP (10). This observation had been interpreted as lack of HPrH15A-Ser46-P binding to CcpA, but the more sensitive SPR measurements conducted here show that the mutated phospho-proteins bind CcpA just like wild type HPr-Ser46-P, but their affinity for CcpA is not enhanced by FBP anymore. Residue Asp-297 of CcpA is the corresponding determinant for the stimulatory effect of FBP and Glc-6-P, since CcpA D297A binds Crh-Ser46-P and HPr-Ser46-P like wild type CcpA, but the affinity to HPr-Ser46-P is no longer increased by FBP or by Glc-6-P. Crh contains a Gln residue at position 15, and binding of Crh-Ser46-P to CcpA or cre is not stimulated by FBP (11). In agreement with the suggested role of His-15, the CrhQ15H mutation makes interaction of CrhQ15HSer46-P with CcpA and cre sensitive for FBP stimulation. Inspection of the crystal structures (16) (17) reveals a difference between the interactions of His-15 in HPr-Ser46-P and Gln-15 in Crh-Ser46-P with CcpA: the latter contacts Arg-325 and not the signal transferring residue Asp-297, which may be the reason for the lack of FBP and Glc-6-P stimulation. In previous in vivo measurements, the HPrH15A mutant displayed only residual CcpA dependent CCR of the gnt operon (32). Since this mutation abolishes exclusively FBP stimulation, this might reflect the influence of FBP on expression of the gnt operon.


Figure 8
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  		FIGURE 8.
Presentation of the contacts of His-15 and Arg-17 of HPr-Ser46-P and of Gln-15 of Crh-Ser46-P in the recently published CcpA-cre complexes (16). A, the ternary complex of CcpA, HPr-Ser46-P, and cre. The monomers of the CcpA dimer are shown in white and gray, the HPr-Ser-46-P proteins in red and orange, and the DNA strands in black and white. HPr-Ser46-P or Crh-Ser46-P bound to CcpA and cre are arranged in line with the DNA. B, contacts of Arg-17 of HPr with residues Asp-84, Asp-69', and Asp-99' in the two N-terminal subdomains of the CcpA dimer. The corresponding residues in B. subtilis CcpA are Asp-85, Asp-70', and Asp-100', respectively. C, residue His-15 of HPr-Ser46-P contacting Asp-296 in CcpA from B. megaterium. The corresponding residue in B. subtilis CcpA is Asp-297. D, residue Gln-15 of Crh-Ser46-P contacting Arg-324 in CcpA from B. megaterium. The corresponding residue in B. subtilis CcpA is Arg-325.

 


Figure 9
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  		FIGURE 9.
Scheme of signal processing by HPr residues His-15 and Arg-17. Left picture, FBP and Glc-6-P enhance phospho-protein binding if Asp-296 of CcpA contacts His-15 of HPr-Ser46-P (horizontal arrows). Right picture, residue Arg-17 of the phospho-proteins interacts with Asp-84, Asp-69', and Asp-99' of the CcpA dimer (horizontal arrow) and participates in the cre binding via positioning of the N-terminal CcpA core subdomains (vertical arrow).

 
In conclusion, we demonstrate here that CcpA is a rather untypical member of the LacI/GalR family of bacterial regulators. While it undergoes similar conformational changes between the DNA binding and non-binding forms like other members of that family, it makes use of two different effectors binding to different sites of the protein to accomplish this task. Binding of a typical LacI/GalR-like effector into the respective binding cleft of CcpA4 leads to an increase of affinity for the phospho-protein effector, which, in turn, triggers the allosterical conformational change necessary for cre binding. The structural sketch shown in Fig. 9 indicates these two modes of signal transduction in CcpA. The detailed functional analysis of the signal transduction mutants in this study reveals molecular details of this untypical mechanism of activation.


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft through SFB 473 and Graduiertenkolleg 805 and the Fonds der Chemis-chen Industrie. 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

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed. Tel.: 49-9131-8528081; Fax: 49-9131-8528082; E-mail: whillen{at}biologie.uni-erlangen.de.

3 The abbreviations used are: CCR, carbon catabolite regulation; FBP, fructose 1,6-bisphosphate; PTS, phosphoenolpyruvate:sugar phosphotransferase system; RU, response unit(s); SPR, surface plasmon resonance. Back

4 M. Schumacher and R. G. Brennan, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Drs. M. Schumacher and R. G. Brennan for communicating unpublished results; Dr. Eva Henssler for kindly providing TetR; Mareen Sprehe for the B. subtilis strains WH490, WH483, and WH487; and Dr. Marco Diel for pWH1543 and Dr. Masayuki Takahashi (Université de Nantes) for helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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