Surface immunolocalisation of HPr in the equine pathogen Streptococcus equi

We have investigated the surface localisation of the phosphotransferase system protein HPr in the equine pathogen Streptococcus equi subsp. equi using immunogold localisation and transmission electron microscopy. Like the LppC acid phosphatase lipoprotein, a reference surface antigen, the S. equi HPR could be clearly detected on the surfaces of intact cells. This study is consistent with previous reports that some streptococcal HPr is cell surface associated and suggests that the extracytoplasmic mobilisation and transfer of phosphate groups by streptococci warrant further investigation.     

HPr as a model protein in structure, interaction, folding and stability studies

The small size and lack of disulphide bonds or cofactors in the Histidine-containing phosphocarrier protein (HPr) makes it an attractive system with which to study structure, interaction to its enzymatic partners, and its stability and folding. Here we give an overview on the immense work that has been performed on this protein and we will show that HPr has been widely used as a model protein to study important aspects in modern Structural Biology.     

Properties of a Streptococcus salivarius spontaneous mutant in which the methionine at position 48 in the protein HPr has been replaced by a valine.

HPr is a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) that participates in the concomitant transport and phosphorylation of sugars in bacteria. In gram-positive bacteria, HPr is also reversibly phosphorylated at a seryl residue at position 46 (Ser-46) by a metabolite-activated ATP-dependent kinase and a Pi-dependent HPr(Ser-P) phosphatase. We report in this article the isolation of a spontaneous mutant (mutant A66) from a streptococcus (Streptococcus salivarius) in which the methionine at position 48 (Met-48) in the protein HPr has been replaced by a valine (Val). The mutation inhibited the phosphorylation of HPr on Ser-46 by the ATP-dependent kinase but did not prevent phosphorylation of HPr by enzyme I or the phosphorylation of enzyme II complexes by HPr(His-P). The results, however, suggested that replacement of Met-48 by Val decreased the affinity of enzyme I for HPr or the affinity of enzyme II proteins for HPr(His-P) or both. Characterization of mutant A66 demonstrated that it has pleiotropic properties, including the lack of IIILman, a specific protein of the mannose PTS; decreased levels of HPr; derepression of some cytoplasmic proteins; reduced growth on PTS as well as on non-PTS sugars; and aberrant growth in medium containing a mixture of sugars.     

Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram-positive bacteria

CcpA, the repressor/activator mediating carbon catabolite repression and glucose activation in many Gram-positive bacteria, has been purified from Bacillus megaterium after fusing it to a His tag. CcpA-his immobilized on a Ni-NTA resin specifically interacted with HPr phosphorylated at seryl residue 46. HPr, a phosphocarrier protein of the phosphoenolpyruvate: glycose phosphotransferase system (PTS), can be phosphorylated at two different sites: (i) at His-15 in a PEP-dependent reaction catalysed by enzyme I of the PTS; and (ii) at Ser-46 in an ATP-dependent reaction catalysed by a metabolite-activated protein kinase. Neither unphosphorylated HPr nor HPr phosphorylated at His-15 nor the doubly phosphorylated HPr bound to CcpA. The interaction with seryl-phosphorylated HPr required the presence of fructose 1,6-bisphosphate. These findings suggest that carbon catabolite repression in Gram-positive bacteria is a protein kinase-triggered mechanism. Glycolytic intermediates, stimulating the corresponding protein kinase and the P-ser-HPr/CcpA complex formation, provide a link between glycolytic activity and carbon catabolite repression. The sensitivity of this complex formation to phosphorylation of HPr at His-15 also suggests a link between carbon catabolite repression and PTS transport activity.     

The phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. 3. 1H and 31P nuclear-magnetic-resonance studies on the phosphocarrier protein HPr; tyrosine titration and denaturation studies.

The phosphocarrier protein HPr has been investigated by proton nuclear magnetic resonance (NMR) at 270 MHz in order to evaluate structural properties of the whole molecule and its active site. The titration behaviour of the three tyrosines of the HPr protein was analysed by monitoring the chemical shifts of the aromatic proton resonances of these residues as a function of pH. It was found that the HPr protein contains a lot of slowly exchanging NH backbone protons which suggested a relatively rigid secondary structure of the protein molecule itself although it contains no disulfide bridges. The HPr protein shows a sharp reversible denaturation behaviour at alkaline pH values. Between pH 10.8 and 11.1 two C-2 proton resonance peaks for the single histidine residue could be observed together with abrupt changes in the aromatic and aliphatic absorption region of the HPr protein which are due to chemical exchange processes. The NMR spectrum of the HPr protein is only changed a little upon raising the temperature from 14 degrees C to 70 degrees C. At 76 degrees C all resonances in the spectrum broaden and almost disappear. This process is irreversible.     

Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system: purification and characterization of the phosphocarrier protein.

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble enzyme I, and a soluble phosphocarrier protein, HPr. The HPr has been purified to homogeneity by a combination of ammonium sulfate precipitations, gel filtration and diethylaminoethyl, carboxymethyl Bio-Gel A, and hydroxylapatite column chromatography. The purified protein is relatively heat stable (ca. 50% activity survives 30 min of boiling) and has a molecular weight of ca. 10,000 (determined by sodium dodecyl sulfate-gel electrophoresis and amino acid analysis). It contains a single histidine residue per molecule and can be totally inactivated by photooxidation with Rose Bengal dye. Although the mycoplasma HPr is very similar to that of Escherichia coli, it shows no significant association with antiserum produced against E. coli HPr.     

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolytic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system, HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction [Deutscher, J., & Saier, M. H., Jr. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6790-6794]. The site of ATP-dependent phosphorylation in HPr of S. faecalis has now been determined. [32P]P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, we obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, we isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of mutant histidine-containing proteins of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli and Salmonella typhimurium.

Histidine-containing phosphocarrier protein (HPr) is common to all of the phosphoenolpyruvate:sugar phosphotransferase systems (PTS) in Escherichia coli and Salmonella typhimurium, except the fructose-specific PTS. Strains which lack HPr activity (ptsH) have been characterized in the past, and it has proved difficult to delineate between tight and leaky mutants. In this study four different parameters of ptsH strains were measured: in vitro sugar phosphorylation activity of the mutant HPr; detection of 32P-labeled P-HPr; ability of monoclonal antibodies to bind mutant HPr; and sensitivity of ptsH strains to fosfomycin. Tight ptsH strains could be defined; they were fosfomycin resistant and produced no HPr protein or completely inactive mutant HPr. All leaky ptsH strains were fosfomycin sensitive, usually produced normal amounts of mutant HPr protein, and had low but measurable activity, and HPr was detectable as a phosphoprotein. This indicates that the regulatory functions of the PTS require a very low level of HPr activity (about 1%). The antibodies used to detect mutant HPr in crude extracts were two monoclonal immunoglobulin G antibodies Jel42 and Jel44. Both antibodies, which have different pIs, inhibited PTS sugar phosphorylation assays, but the antibody-HPr complex could still be phosphorylated by enzyme I. Preliminary evidence suggests that the antibodies bind to two different epitopes which are in part located in a beta-sheet structure.     

The galU gene expression in Streptococcus pneumoniae

Bacillus subtilis possesses carbon-flux regulating histidine protein (Crh), a paralog of the histidine protein (HPr) of the phosphotransferase system (PTS). Like HPr, Crh becomes (de)phosphorylated in vitro at residue Ser46 by the metabolite-controlled HPr kinase/phosphorylase HPrK/P. Depending on its phosphorylation state, Crh exerts regulatory functions in connection with carbohydrate metabolism. So far, knowledge on phosphorylation of Crh in vivo has been limited and derived from indirect evidence. Here, we studied the dynamics of Crh phosphorylation directly by non-denaturing gel electrophoresis followed by Western analysis. The results confirm that HPrK/P is the single kinase catalyzing phosphorylation of Crh in vivo. Accordingly, phosphorylation of Crh is triggered by the carbon source as observed previously for HPr, but with some differences. Phosphorylation of both proteins occurred during exponential growth and disappeared upon exhaustion of the carbon source. During exponential growth, ~80% of the Crh molecules were phosphorylated when cells utilized a preferred carbon source. The reverse distribution, i.e. around 20% of Crh molecules phosphorylated, was obtained upon utilization of less favorable substrates. This clear-cut classification of the substrates into two groups has not previously been observed for HPr(Ser)~P formation. The likely reason for this difference is the additional PTS-dependent phosphorylation of HPr at His15, which limits accumulation of HPr(Ser)~P.     

The ptsH gene from Bacillus thuringiensis israelensis. Characterization of a new phosphorylation site on the protein HPr.

The ptsH gene from Bacillus thuringiensis israelensis (Bti), coding for the phosphocarrier protein HPr of the phosphotransferase system has been cloned and overexpressed in Escherichia coli. Comparison of its primary sequence with other HPr sequences revealed that the conserved His15 and Ser46 residues were shifted by one amino acid and located at positions 14 and 45, respectively. The biological activity of the protein was not affected by this change. When expressed in a Bacillus subtilis ptsH deletion strain, Bti HPr was able to complement the functions of HPr in sugar uptake and glucose catabolite repression of the gnt and iol operons. A modified form of HPr was detected in Bti cells, and also when Bti ptsH was expressed in E. coli or B. subtilis. This modification was identified as phosphorylation, because alkaline phosphatase treatment converted the modified form to unmodified HPr. The phosphoryl bond in the new form of in vivo phosphorylated HPr was resistant to alkali treatment but sensitive to acid treatment, suggesting phosphorylation at a histidine residue. Replacement of His14 with alanine in Bti HPr prevented formation of the new form of phosphorylated HPr. The phosphorylated HPr was stable at 60 degrees C, in contrast with HPr phosphorylated at the N delta 1 position of His14 with phosphoenolpyruvate and enzyme I. (31)P-NMR spectroscopy was used to show that the new form of P-HPr carried the phosphoryl group bound to the N epsilon 2 position of His14 of Bti HPr. Phosphorylation of HPr at the novel site did not occur when Bti HPr was expressed in an enzyme I-deficient B. subtilis strain. In addition, P-(N epsilon 2)His-HPr did not transfer its phosphoryl group to the purified glucose-specific enzyme IIA domain of B. subtilis.     

Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 1. Sequential resonance assignments

Two-dimensional NMR studies at 500 MHz have been performed on the histidine-containing protein (HPr) from Escherichia coli. HPr is one of the phosphocarrier proteins involved in the bacterial phosphoenolpyruvate:sugar phosphotransferase system that is responsible for the concomitant phosphorylation and translocation of a number of sugars. Sequential resonance assignments of HPr are complete. The conventional method of sequential assignments involving J-correlated spectroscopy (COSY) and nuclear Overhauser spectroscopy (NOESY) has been supplemented by optimized relayed coherence transfer spectroscopy (RELAY) to help overcome the spectral overlap that is inevitable in the spectra of proteins the size of HPr. RELAY experiments were performed in H2O to obtain NH-C beta H connectivities and in D2O to obtain C alpha H-C gamma H connectivities. The abundance of relayed coherence transfer peaks in the two experiments greatly aided in the assignment process of the complicated protein spectrum. The assignments lay the groundwork for the determination of the solution structure of HPr, as described in the accompanying paper [Klevit, R. E., & Waygood, E. B. (1986) Biochemistry (third paper of three in this issue)].     

Carbon catabolite repression by the catabolite control protein CcpA in Staphylococcus xylosus

Carbon catabolic repression (CR) by the catabolite control protein CcpA has been analyzed in Staphylococcus xylosus. Genes encoding components needed to utilize lactose, sucrose, and maltose were found to be repressed by CcpA. In addition, the ccpA gene is under negative autogenous control. Among several tested sugars, glucose caused strongest CcpA-dependent repression. Glucose can enter S. xylosus in nonphosphorylated form via the glucose uptake protein GlcU. Internal glucose is then phosphorylated by the glucose kinase GlkA. Alternatively, glucose can be transported and concomitantly phosphorylated by glucose-specific permease(s) of the phosphotransferase system (PTS). S. xylosus mutant strains deficient in GlcU or GlkA showed partial relief of glucose-specific, CcpA-dependent repression. Likewise, blocking PTS activity completely by inactivation of the gene encoding the general PTS protein enzyme I resulted in diminished glucose-mediated repression. Thus, both glucose entry routes contribute to glucose-specific CR in S. xylosus. The sugar transport activity of the PTS is not required to trigger glucose-specific repression. The phosphocarrier protein HPr however, is absolutely essential for CcpA activity. Inactivation of the HPr gene led to a complete loss of CR. Repression is also abolished upon inactivation of the HPr kinase gene or by replacing serine at position 46 of HPr by alanine. These results clearly show that HPr kinase provides the signal, seryl-phosphorylated HPr, to activate CcpA in S. xylosus.     

Purification and characterization of an ATP-dependent protein kinase from Streptococcus faecalis

Abstract An enzyme catalyzing the ATP-dependent phosphorylation of HPr of the bacterial phosphotransferase system has been purified from Streptococcus faecalis . Size exclusion chromatography and sodium dodecyl sulfate (SDS) polyacrylamide gels revealed an M _r of 65000. Beside HPr of S. faecalis the protein kinase also phosphorylates HPr of Streptococcus lactis, Streptococcus pyogenes, Bacillus subtilis and Streptococcus aureus , but not HPr of Escherichia coli . The kinase is largely inhibited by P_i and EDTA. Mg²⁺ and Mn²⁺ could overcome inhibition by EDTA. 2-Phosphoglycerate and glucose-6-phosphate, previously reported to stimulate kinase activity in crude extracts, had no effect on the purified enzyme. Fructose-1,6-diphosphate stimulated the protein kinase.     

The histidine-phosphocarrier protein of Streptomyces coelicolor folds by a partially folded species at low pH.

The folding of a 93-residue protein, the histidine-phosphocarrier protein of Streptomyces coelicolor, HPr, has been studied using several biophysical techniques, namely fluorescence, 8-anilinonaphthalene-1-sulfate binding, circular dichroism, Fourier transform infrared spectroscopy, gel filtration chromatography and differential scanning calorimetry. The chemical-denaturation behaviour of HPr, followed by fluorescence, CD and gel filtration, at pH 7.5 and 25 degrees C, is described as a two-state process, which does not involve the accumulation of thermodynamically stable intermediates. Its conformational stability under those conditions is deltaG = 4.0 +/- 0.2 kcal x mol-1 (1 kcal = 4.18 kJ), which makes the HPr from S. coelicolor the most unstable member of the HPr family described so far. The stability of the protein does not change significantly from pH 7-9, as concluded from the differential scanning calorimetry and thermal CD experiments. Conformational studies at low pH (pH 2.5-4) suggest that, in the absence of cosmotropic agents, HPr does not unfold completely; rather, it accumulates partially folded species. The transition from those species to other states with native-like secondary and tertiary structure, occurs with a pKa = 3.3 +/- 0.3, as measured by the averaged measurements obtained by CD and fluorescence. However, this transition does not agree either with: (a) that measured by burial of hydrophobic patches (8-anilinonaphthalene-1-sulfate binding experiments); or (b) that measured by acquisition of native-like compactness (gel-filtration studies). It seems that acquisition of native-like features occurs in a wide pH range and it cannot be ascribed to a unique side-chain titration. These series of intermediates have not been reported previously in any member of the HPr family.     

The HPr proteins from the thermophile Bacillus stearothermophilus can form domain-swapped dimers

The study of proteins from extremophilic organisms continues to generate interest in the field of protein folding because paradigms explaining the enhanced stability of these proteins still elude us and such studies have the potential to further our knowledge of the forces stabilizing proteins. We have undertaken such a study with our model protein HPr from a mesophile, Bacillus subtilis, and a thermophile, Bacillus stearothermophilus. We report here the high-resolution structures of the wild-type HPr protein from the thermophile and a variant, F29W. The variant proved to crystallize in two forms: a monomeric form with a structure very similar to the wild-type protein as well as a domain-swapped dimer. Interestingly, the structure of the domain-swapped dimer for HPr is very different from that observed for a homologous protein, Crh, from B.subtilis. The existence of a domain-swapped dimer has implications for amyloid formation and is consistent with recent results showing that the HPr proteins can form amyloid fibrils. We also characterized the conformational stability of the thermophilic HPr proteins using thermal and solvent denaturation methods and have used the high-resolution structures in an attempt to explain the differences in stability between the different HPr proteins. Finally, we present a detailed analysis of the solution properties of the HPr proteins using a variety of biochemical and biophysical methods.     

HPr polymorphism in oral streptococci is caused by the partial removal of the N-terminal methionine.

HPr is a small phosphocarrier protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system involved in the transport and phosphorylation of sugars. It has recently been reported that streptococci possess two forms of HPr having identical biochemical properties. In this communication, we show by N-terminal amino-acid sequencing and by ion-spray mass spectroscopy that these two forms differ by the presence or the absence of the N-terminal methionine.     

Solution structure and dynamics of Crh, the Bacillus subtilis catabolite repression HPr

The solution structure and dynamics of the Bacillus subtilis HPr-like protein, Crh, have been investigated using NMR spectroscopy. Crh exhibits high sequence identity (45 %) to the histidine-containing protein (HPr), a phospho-carrier protein of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system, but contains no catalytic His15, the site of PEP-dependent phosphorylation in HPr. Crh also forms a mixture of monomers and dimers in solution whereas HPr is known to be monomeric. Complete backbone and side-chain assignments were obtained for the monomeric form, and 60 % of the dimer backbone resonances; allowing the identification of the Crh dimer interface from chemical-shift mapping. The conformation of Crh was determined to a precision of 0.46(+/-0.06) A for the backbone atoms, and 1.01(+/-0.08) A for the heavy atoms. The monomer structure is similar to that of known HPr 2.67(+/-0.22) A (C(alpha) rmsd), but has a few notable differences, including a change in the orientation of one of the helices (B), and a two-residue shift in beta-sheet pairing of the N-terminal strand with the beta4 strand. This shift results in a shortening of the surface loop present in HPr and consequently provides a flatter surface in the region of dimerisation contact, which may be related to the different oligomeric nature of these two proteins. A binding site of phospho-serine(P-Ser)-Crh with catabolite control protein A (CcpA) is proposed on the basis of highly conserved surface side-chains between Crh and HPr. This binding site is consistent with the model of a dimer-dimer interaction between P-Ser-Crh and CcpA. (15)N relaxation measured in the monomeric form also identified differential local mobility in the helix B which is located in the vicinity of this site.     

Catabolite repression resistance of gnt operon expression in Bacillus subtilis conferred by mutation of His-15, the site of phosphoenolpyruvate-dependent phosphorylation of the phosphocarrier protein HPr.

Carbon catabolite repression of the gnt operon of Bacillus subtilis is mediated by the catabolite control protein CcpA and by HPr, a phosphocarrier protein of the phosphotransferase system. ATP-dependent phosphorylation of HPr at Ser-46 is required for carbon catabolite repression as ptsH1 mutants in which Ser-46 of HPr is replaced with an unphosphorylatable alanyl residue are resistant to carbon catabolite repression. We here demonstrate that mutation of His-15 of HPr, the site of phosphoenolpyruvate-dependent phosphorylation, also prevents carbon catabolite repression of the gnt operon. A strain which expressed two mutant HPrs (one in which Ser-46 is replaced by Ala [S46A HPr] and one in which His-15 is replaced by Ala [H15A HPr]) on the chromosome was barely sensitive to carbon catabolite repression, although the H15A mutant HPr can be phosphorylated at Ser-46 by the ATP-dependent HPr kinase in vitro and in vivo. The S46D mutant HPr which structurally resembles seryl-phosphorylated HPr has a repressive effect on gnt expression even in the absence of a repressing sugar. By contrast, the doubly mutated H15E,S46D HPr, which resembles the doubly phosphorylated HPr because of the negative charges introduced by the mutations at both phosphorylation sites, had no such effect. In vitro assays substantiated these findings and demonstrated that in contrast to the wild-type seryl-phosphorylated HPr and the S46D mutant HPr, seryl-phosphorylated H15A mutant HPr and H15E,S46D doubly mutated HPr did not interact with CcpA. These results suggest that His-15 of HPr is important for carbon catabolite repression and that either mutation or phosphorylation at His-15 can prevent carbon catabolite repression.     

The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon.

The LevR protein is the activator of expression of the levanase operon of Bacillus subtilis. The promoter of this operon is recognized by RNA polymerase containing the sigma 54-like factor sigma L. One domain of the LevR protein is homologous to activators of the NtrC family, and another resembles antiterminator proteins of the BglG family. It has been proposed that the domain which is similar to antiterminators is a target of phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent regulation of LevR activity. We show that the LevR protein is not only negatively regulated by the fructose-specific enzyme IIA/B of the phosphotransferase system encoded by the levanase operon (lev-PTS) but also positively controlled by the histidine-containing phosphocarrier protein (HPr) of the PTS. This second type of control of LevR activity depends on phosphoenolpyruvate-dependent phosphorylation of HPr histidine 15, as demonstrated with point mutations in the ptsH gene encoding HPr. In vitro phosphorylation of partially purified LevR was obtained in the presence of phosphoenolpyruvate, enzyme I, and HPr. The dependence of truncated LevR polypeptides on stimulation by HPr indicated that the domain homologous to antiterminators is the target of HPr-dependent regulation of LevR activity. This domain appears to be duplicated in the LevR protein. The first antiterminator-like domain seems to be the target of enzyme I and HPr-dependent phosphorylation and the site of LevR activation, whereas the carboxy-terminal antiterminator-like domain could be the target for negative regulation by the lev-PTS.