Substitution of aspartate and glutamate for active center histidines in the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system maintain phosphotransfer potential.

The active center histidines of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system proteins; histidine-containing protein, enzyme I, and enzyme IIA(Glc) were substituted with a series of amino acids (serine, threonine, tyrosine, cysteine, aspartate, and glutamate) with the potential to undergo phosphorylation. The mutants [H189E]enzyme I, [H15D]HPr, and [H90E]enzyme IIA(Glc) retained ability for phosphorylation as indicated by [(32)P]phosphoenolpyruvate labeling. As the active center histidines of both enzyme I and enzyme IIA(Glc) undergo phosphorylation of the N(epsilon2) atom, while HPr is phosphorylated at the N(delta1) atom, a pattern of successful substitution of glutamates for N(epsilon2) phosphorylations and aspartates for N(delta1) phosphorylations emerges. Furthermore, phosphotransfer between acyl residues: P-aspartyl to glutamyl and P-glutamyl to aspartyl was demonstrated with these mutant proteins and enzymes.

Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system. In vitro intragenic complementation: the roles of Arg126 in phosphoryl transfer and the C-terminal domain in dimerization.

Enzyme I mutants of the Salmonella typhimurium phosphoenolpyruvate:sugar phosphotransferase system (PTS), which show in vitro intragenic complementation, have been identified as Arg126Cys (strain SB1690 ptsI34), Gly356Ser (strain SB1681 ptsI16), and Arg375Cys (strain SB1476 ptsI17). The mutation Arg126Cys is in the N-terminal HPr-binding domain, and complements Gly356Ser and Arg375Cys enzyme I mutations located in the C-terminal phosphoenolpyruvate(PEP)-binding domain. Complementation results in the formation of unstable heterodimers. None of the mutations alters the K(m) for HPr, which is phosphorylated by enzyme I. Arg126 is a conserved residue; the Arg126Cys mutation gives a V(max) of 0.04% wild-type, establishing a role in phosphoryl transfer. The Gly356Ser and Arg375Cys mutations reduce enzyme I V(max) to 4 and 2%, respectively, and for both, the PEP K(m) is increased from 0.1 to 3 mM. It is concluded that this activity was from the monomer, rather than the dimer normally found in assays of wild-type. In the presence of Arg126Cys enzyme, V(max) for Gly356Ser and Arg375Cys enzymes I increased 6- and 2-fold, respectively; the K(m) for PEP decreased to <10 microM, but the K(m) became dependent upon the stability of the heterodimer in the assay. Gly356 is conserved in enzyme I and pyruvate phosphate dikinase, which is a homologue of enzyme I, and this residue is part of a conserved sequence in the subunit interaction site. Gly356Ser mutation impairs enzyme I dimerization. The mutation Arg375Cys also impairs dimerization, but the equivalent residue in pyruvate phosphate dikinase is not associated with the subunit interaction site. A 37 000 Da, C-terminal domain of enzyme I has been expressed and purified; it dimerizes and complements Gly356Ser and Arg375Cys enzymes I proving that the association/dissociation properties of enzyme I are a function of the C-terminal domain.

Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs.

The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding domain (Fv) has been produced as a single chain Fv (scFv). The scFv gene was synthesized in vitro and coded for pelB leader peptide-heavy chain-linker-light chain-(His)(5) tail. The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II. This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352. The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10-30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purified by denaturation/renaturation yielding preparations with K(d) values from 20 to 175 nM. However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 +/- 2.0 nM compared with 2.8 +/- 1.6 and 3.7 +/- 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively. The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was concluded that the small percentage ( approximately 6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay.

The aspartyl replacement of the active site histidine in histidine-containing protein, HPr, of the Escherichia coli Phosphoenolpyruvate:Sugar phosphotransferase system can accept and donate a phosphoryl group. Spontaneous dephosphorylation of acyl-phosphate autocatalyzes an internal cyclization.

The active site residue, His(15), in histidine-containing protein, HPr, can be replaced by aspartate and still act as a phosphoacceptor and phosphodonor with enzyme I and enzyme IIA(glucose), respectively. Other substitutions, including cysteine, glutamate, serine, threonine, and tyrosine, failed to show any activity. Enzyme I K(m) for His(15) --> Asp HPr is increased 10-fold and V(max) is decreased 1000-fold compared with wild type HPr. The phosphorylation of Asp(15) led to a spontaneous internal rearrangement involving the loss of the phosphoryl group and a water molecule, which was confirmed by mass spectrometry. The protein species formed had a higher pI than His(15) --> Asp HPr, which could arise from the formation of a succinimide or an isoimide. Hydrolysis of the isolated high pI form gave only aspartic acid at residue 15, and no isoaspartic acid was detected. This indicates that an isoimide rather than a succinimide is formed. In the absence of phosphorylation, no formation of the high pI form could be found, indicating that phosphorylation catalyzed the formation of the cyclization. The possible involvement of Asn(12) in an internal cyclization with Asp(15) was eliminated by the Asn(12) --> Ala mutation in His(15) --> AspHPr. Asn(12) substitutions of alanine, aspartate, serine, and threonine in wild type HPr indicated a general requirement for residues capable of forming a hydrogen bond with the Nepsilon(2) atom of His(15), but elimination of the hydrogen bond has only a 4-fold decrease in k(cat)/K(m).

Identification of the Escherichia coli enzyme I binding site in histidine-containing protein, HPr, by the effects of mutagenesis.

The structure of the N-terminal domain of enzyme I complexed with histidine-containing protein (HPr) has been described by multi-dimensional NMR. Residues in HPr involved in binding were identified by intermolecular nuclear Overhauser effects (Garrett et al. 1999). Most of these residues have been mutated, and the effect of these changes on binding has been assessed by enzyme I kinetic measurement. Changes to Thr16, Arg17, Lys24, Lys27, Ser46, Leu47, Lys49, Gln51, and Thr56 result in increases to the HPr Km of enzyme I, which would be compatible with changes in binding. Except for mutations to His15 and Arg17, very little or no change in Vmax was found. Alanine replacements for Gln21, Thr52, and Leu55 have no effect. The mutation Lys40Ala also affects HPr Km of enzyme I; residue 40 is contiguous with the enzyme I binding site in HPr and was not identified by NMR. The mutations leading to a reduction in the size of the side chain (Thr16Ala, Arg17Gly, Lys24Ala, Lys27Ala, and Lys49Gly) caused relatively large increases in Km (>5-fold) indicating these residues have more significant roles in binding to enzyme I. Acidic replacement at Ser46 caused very large increases (>100-fold), while Gln51Glu gave a 3-fold increase in Km. While these results essentially concur with the identification of residues by the NMR experiments, the apparent importance of individual residues as determined by mutation and kinetic measurement does not necessarily correspond with the number of contacts derived from observed intermolecular nuclear Overhauser effects.

Determination of the binding constants for three HPr-specific monoclonal antibodies and their Fab fragments.

Jel42, Jel44 and Jel323 are mouse monoclonal antibodies specific for HPr, the histidine-containing phosphocarrier protein, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system. The binding constants, Kd, of the three antibodies and their Fab fragments have been determined: Jel42, 2.8+/-1.6 nM; Jel42Fab, 3. 7+/-0.3 nM; Jel44, 5.1+/-0.4 nM; Jel44Fab, 6.3+/-1.1 nM; Jel323, 5. 7+/-0.5 nM; Jel323Fab, 5.1+/-0.9 nM. The binding constants were determined by a fluorescence polarization assay that used the mutants Arg17Cys HPr and Phe2Cys HPr specifically labeled with fluorescein-5-maleimide. The latter was used for Jel323 as interaction with fluorescein-5-maleimide-labeled Arg17Cys HPr gave quenching of the fluorescence intensity. The specificity of each antibody and the Fab fragments for binding to many HPr mutants was determined by this solution assay. The Fab fragments had the same specificity or cross-reactivity as the antibodies. Comparison of relative binding specificity determined by a solid phase assay showed that the results from both types of assay are comparable. Neither Jel42 nor Jel323 binding was affected by ionic strength (approximately 45 to 245 mM salt), but Jel44 varied about two- to threefold. Charged residues are prominent in the Jel44 epitope and paratope. Initial thermodynamic characterization was investigated by temperature-dependent determinations of the Kd. The binding of Jel42 and Jel323 to HPr was entropic at low temperatures and enthalpic at physiological temperatures. Jel44 showed no change in the contributions of entropy and enthalpy over the temperature range 3 to 37 degreesC. The 2.5 A resolution structure of the complex of Jel42 Fab fragment bound to HPr described in the accompanying paper provides some structural intepretation for the mutational effects.

The 2.5 A resolution structure of the jel42 Fab fragment/HPr complex.

The tertiary structure of Jel42 Fab fragment complexed with HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli, has been determined at 2.5 A resolution. X-ray diffraction from a larger crystal provided 22,067 unique reflections as compared to 14,763 unique reflections (2.8 A resolution), which were obtained previously from a smaller crystal. The higher resolution allowed for more precise location of amino acid side-chains and for the location of well-ordered water molecules. Five more residues in the Fab fragment are found to be involved in binding HPr and two additional residues are identified as part of the epitope, bringing the totals to 24 and 16, respectively. At least nine water molecules are found at the interface between the two proteins, and these mediate hydrogen bonding interactions between the Fab fragment and HPr. Three additional hydrogen bonds have been identified (bringing the total to ten) and one salt-bridge occurs between LysL50 of the L2 complementarity-determining region (CDR) and GluP66 of HPr. This salt-bridge is the only interaction between HPr and CDRL2; thus all six CDRs are involved in binding. Inspection and empirical energy minimization of mutant HPrs in the complex indicate that, in some cases in the binding interaction, water molecules may compensate for residue alterations. Binding to the mutant SerP64Tyr HPr may require a movement of the HPr main chain. The active centre region of HPr, which is not involved in binding the antibody, and which was not resolved in the 2.8 A resolution structure of the complex, was determined. This active centre determined at pH 5.8, which is completely free of intermolecular contacts due to crystal packing, shows a potential hydrogen bond between the AsnP12 OD1 atom and the HisP15 NE2 atom, and no involvement of the C terminus with HisP15. The HisP15 ND1 atom is the site of phosphorylation in HPr. Although a specific amino acid at residue 12 is not conserved in HPr molecules from all species, a hydrogen bond between the side-chains of residue 12 and HisP15 may be a conserved feature of the active centres.

The structure and function of HPr.

Histidine-containing phosphocarrier protein, HPr, was one of the early protein tertiary structures determined by two-dimensional 1H-NMR. Tertiary structures for HPrs from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus have been obtained by 1H NMR and the overall folding pattern of HPr is highly conserved, a betaalpha betabeta alphabeta alpha arrangement of three alpha-helices overlaying a four-stranded beta-sheet. High-resolution structures for HPrs from E. coli and B. subtilis have been obtained using 15N- and 13C-labeled proteins. The first application of NMR to the understanding of the structure and function of HPr was to describe the phosphohistidine isomer, Ndelta1-P-histidine in S. aureus phospho-HPr, and the unusual pKas of the His-15 side chain. The pKa values for the His-15 imidazole from more recent studies are 5.4 for HPr and 7.8 for phospho-HPr from E. coli, for example. A consensus description of the active site is proposed for HPr and phospho-HPr. In HPr, His-15 has a defined conformation and N-caps helix A, and is thus affected by the helix dipole. His-15 undergoes a small conformational change upon phosphorylation, a movement to allow the phosphoryl group to be positioned such that it forms hydrogen bonds with the main chain amide nitrogens of residue 16 (not conserved) and Arg-17. Interactions between residue 12 side chain (not conserved: asparagine, serine, and threonine) and His-15, and between the Arg-17 guanidinium group and the phosphoryl group, are either weak or transitory.

Conversion of an antibody into an enzyme which cleaves the protein HPr.

Jel 42 is an IgG which binds to the small bacterial protein, HPr and the structure of the complex is known at high resolution. The IgG was expressed as a single chain variable fragment (scFv) and the binding to HPr was assessed by fluorescence polarization of fluorescein-labelled HPr. The binding constant for the IgG was about 20-fold higher than the scFv. Inspection of the structure of the complex suggested that it might be possible to convert the scFv into a bond-specific protease by the introduction of three catalytic residues: a glutamate to increase the nucleophilicity of a nearby water molecule, a lysine to increase the polarizability of the carbonyl group and a histidine to provide a proton to convert the amine into a better leaving group. By trial and error it was found that a fourth residue had to be converted into glycine in order to maintain the integrity of complimentarity-determining region three of the heavy chain (CDRH3) at the binding interface. The resulting quadruple mutant still bound to HPr and unlike other mutants, showed weak protease activity as judged from the fluorescence polarization assay. The activity was maximum at pH 6 consistent with a requirement for a protonated histidine residue. With the aid of HPr fluorescein-labelled at two different positions, it was demonstrated that the size of the products was consistent with cleavage occurring in the vicinity of the target peptide bond. The activity was specific for HPr since an excess of bovine serum albumin did not interfere with the reaction.

Demonstration of protein-protein interaction specificity by NMR chemical shift mapping.

Chemical shift mapping is becoming a popular method for studying protein-protein interactions in solution. The technique is used to identify putative sites of interaction on a protein surface by detecting chemical shift perturbations in simple (1H, 15N)-HSQC NMR spectra of a uniformly labeled protein as a function of added (unlabeled) target protein. The high concentrations required for these experiments raise questions concerning the possibility for non-specific interactions being detected, thereby compromising the information obtained. We demonstrate here that the simple chemical shift mapping approach faithfully reproduces the known functional specificities among pairs of closely related proteins from the phosphoenolpyruvate:sugar phosphotransferase systems of Escherichia coli and Bacillus subtilis.

Mutation of serine-46 to aspartate in the histidine-containing protein of Escherichia coli mimics the inactivation by phosphorylation of serine-46 in HPrs from gram-positive bacteria.

Histidine-containing protein (HPr) is a phosphocarrier protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. HPr is phosphorylated at the active site residue, His15, by phosphoenolpyruvate-dependent enzyme I in the first enzyme reaction in the process of phosphoryl transfer to sugar. In many Gram-positive bacterial species HPr may also be phosphorylated at Ser46 by an ATP-dependent protein kinase but not in the Gram-negative Escherichia coli and Salmonella typhimurium. One effect of the phosphorylation at Ser46 is to make HPr a poor acceptor for phosphorylation at His15. In Bacillus subtilis HPr, the mutation Ser46Asp mimics the effects of phosphorylation. A series of mutations were made at Ser46 in E. coli HPr: Ala, Arg, Asn, Asp, Glu, and Gly. The two acidic replacements mimic the effects of phosphorylation of Ser46 in HPrs from Gram-positive bacteria. In particular, when mutated to Asp46, the His 15 phosphoacceptor activity (enzyme I Km/Kcat) decreases by about 2000-fold (enzyme I Km, 4 mM HPr; Kcat, approximately 30%). The alanine and glycine mutations had near-wild-type properties, and the asparagine and arginine mutations yielded small changes to the Km values. The crystallographic tertiary structure of Ser46Asp HPr has been determined at 1.5 A resolution, and several changes have been observed which appear to be the effect of the mutation. There is a tightening of helix B, which is demonstrated by a consistent shortening of hydrogen bond lengths throughout the helix as compared to the wild-type structure. There is a repositioning of the Gly54 residue to adopt a 3(10) helical pattern which is not present in the wild-type HPr. In addition, the higher resolution of the mutant structure allows for a more definitive placement of the carbonyl of Pro11. The consequence of this change is that there is no torsion angle strain at residue 16. This result suggests that there is no active site torsion angle strain in wild-type E. coli HPr. The lack of substantial change at the active center of E. coli HPr Ser46Asp HPr suggests that the effect of the Ser46 phosphorylation in HPrs from Gram-positive bacteria is due to an electrostatic interference with enzyme I binding.

Influence of N-cap mutations on the structure and stability of Escherichia coli HPr.

This paper describes the effect of N-capping substitutions on the structure and stability of histidine-containing protein (HPr). We have used NMR spectroscopy and conformational stability studies to quantify changes in local and global free energy due to mutagenesis at Ser46, the N-cap for helix B in HPr. Previous NMR studies suggested that helix B of Escherichia coli HPr is dynamic as judged by the rate of exchange of amide protons with solvent. Ser46 was chosen because it is the site of regulatory phosphorylation in HPrs from Gram-positive bacteria, and mutation of this residue to an aspartic acid (S46D) in E. coli HPr (Gram-negative) also makes it a poor substrate in the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Therefore, to understand the mechanism of inactivation of E. coli S46D HPr, as well as the effect of mutagenesis on protein stability, we have characterized three mutants of E. coli HPr: Ser46 has been mutated to an Asp, Asn, and Ala in S46D, S46N, and S46A HPrs, respectively. The results indicate that these N-cap replacements have a marked influence on helix B stability. The effect of mutagenesis on local stability is correlated to global unfolding of HPr. The ability of amino acids to stabilize helix B is Asp > Asn > Ser > Ala. In addition, since there are neither large-scale conformational changes nor detectable changes in the active site of S46D HPr, it is proposed that the loss of phosphotransfer activity of S46D HPr is due to unfavorable steric and/or electrostatic interactions of the Asp with enzyme I of the PTS.

The Escherichia coli adenylyl cyclase complex: requirement of PTS proteins for stimulation by nucleotides.

GTP, as well as other nucleoside triphosphates, stimulates the activity of Escherichia coli adenylyl cyclase in permeable cells; the stimulatory effect is lost when the cells are disrupted by passage through a French pressure cell. These data suggested that the allosteric regulation by GTP of adenylyl cyclase activity requires an interaction of the enzyme with other protein factors. Strains deleted for genes encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) failed to show an activity stimulation by GTP. With a view to localizing the site of interaction of GTP with the adenylyl cyclase complex, a variety of studies using purified PTS proteins were performed using the photoaffinity labeling reagent, 8-azidoGTP. These studies showed that 8-azidoGTP bound specifically to HPr. A species specificity study showed that the photoaffinity reagent labeled E. coli HPr but not HPr proteins from Mycoplasma capricolum or Bacillus subtilis. A variety of site-directed mutations of E. coli HPr were evaluated for interaction with GTP by photoaffinity labeling as well as by nuclear magnetic resonance; the results of these studies indicate that the lysine residues at positions 24 and 27, serine-46, the threonine at position 36, and the aspartate at position 69 are important for the binding of GTP to HPr. Molecular modeling has been used to formulate a model for the binding of GTP to HPr involving electrostatic interaction of the phosphate groups of the nucleotide with the side chains of lysine residues 27 and 45 and serine-43, interaction of the sugar with serine-46, and interaction of the base with lysine-24. From these data, it is hypothesized that the binding of GTP to HPr is required for the GTP-dependent stimulation of the activity of the adenylyl cyclase complex.

Investigation of a side-chain-side-chain hydrogen bond by mutagenesis, thermodynamics, and NMR spectroscopy.

Anomalous NMR behavior of the hydroxyl proton resonance for Ser 31 has been reported for histidine-containing protein (HPr) from two microorganisms: Escherichia coli and Staphylococcus aureus. The unusual slow exchange and chemical shift exhibited by the resonance led to the proposal that the hydroxyl group is involved in a strong hydrogen bond. To test this hypothesis and to characterize the importance of such an interaction, a mutant in which Ser 31 is replaced by an alanine was generated in HPr from Escherichia coli. The activity, stability, and structure of the mutant HPr were assessed using a reconstituted assay system, analysis of solvent denaturation curves, and NMR, respectively. Substitution of Ser 31 yields a fully functional protein that is only slightly less stable (delta delta G(folding) = 0.46 +/- 0.15 kcal mol-1) than the wild type. The NMR results confirm the identity of the hydrogen bond acceptor as Asp 69 and reveal that it exists as the gauche- conformer in wild-type HPr in solution but exhibits conformational averaging in the mutant protein. The side chain of Asp 69 interacts with two main-chain amide proteins in addition to its interaction with the side chain of Ser 31 in the wild-type protein. These results indicate that removal of the serine has led to the loss of all three hydrogen bond interactions involving Asp 69, suggesting a cooperative network of interactions. A complete analysis of the thermodynamics was performed in which differences in side-chain hydrophobicity and conformational entropy between the two proteins are accounted for.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural consequences of histidine phosphorylation: NMR characterization of the phosphohistidine form of histidine-containing protein from Bacillus subtilis and Escherichia coli.

The bacterial phosphoenolpyruvate:sugar phosphotransferase system involves a series of reactions in which phosphoprotein intermediates are formed. Histidine-containing protein (HPr) is phosphorylated on the N delta 1 position of the imidazole ring of His15 by enzyme I and acts as a phosphoryl donor to the sugar-specific enzymes IIA. The structure of phosphorylated HPr from Bacillus subtilis, primarily, and from Escherichia coli has been studied by nuclear magnetic resonance (NMR) spectroscopy. Phosphorylation of His15 results in large downfield shifts in amide proton and nitrogen resonances for residues 16 and 17 but results in only modest or no shifts in other backbone resonances. The exchange rates of these two amide groups are decreased more than 10-fold upon phosphorylation. Analysis of the coupling constants 3JNH alpha revealed no significant changes throughout the protein, indicating that backbone phi dihedral angles do not change detectably. 3J alpha beta and 3JN beta patterns determined from P.E.COSY and HNHB spectra, respectively, revealed a change in one side chain, that of conserved Arg17. Analysis of NOESY spectra revealed a limited number of changes in NOEs involving protons in Ser12, His15, Arg71, and Pro18 in B. subtilis HPr. The NMR results indicate that the Arg17 side chain becomes limited in its conformational range in the phosphorylated protein, taking on a conformation that points its guanidinium group toward the phosphoryl group on His15. In addition, the tautomeric and ionization states of His15 have been determined using 15N and 31P NMR. At neutral pH, the imidazole is predominantly in the protonated form and the phosphoryl group is in the dianionic form in P-His15. Altogether, the results indicate that phosphorylation of His15 yields only a local effect on the protein's structure. The data are consistent with a small change in the disposition of the histidine side chain, allowing phosphoryl group oxygens to serve as hydrogen bond acceptors for the amide protons of residues Ala16 and Arg17, which constitute the first two residues of an alpha-helix. Thus, similar to many proteins that bind phosphoryl moieties noncovalently, the phosphoryl group in P-His15-HPr is situated to allow for a favorable electrostatic interaction at the N-terminal end of an alpha-helix.

Repair of spontaneously deamidated HPr phosphocarrier protein catalyzed by the L-isoaspartate-(D-aspartate) O-methyltransferase.

The non-enzymatic deamidation at residues Asn-12 and Asn-38 of Escherichia coli phosphocarrier protein, HPr, and the repair of the resulting L-isoaspartyl (or beta-aspartyl) derivatives, HPr-1 and HPr-2, by recombinant human S-adenosylmethionine-dependent L-isoaspartate-(D-aspartate) O-methyltransferase (EC 2.1.1.77) were investigated. HPr is a component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system that is involved in the concomitant translocation and phosphorylation of many hexose sugars. The major products of the deamidation reaction, L-isoaspartyl (or beta-aspartyl) residues at positions 12 and 38, were found to be substrates for the L-isoaspartate-(D-aspartate) O-methyltransferase, an enzyme active on a wide variety of peptides and proteins containing these abnormal residues. This enzyme has been shown to catalyze the first step in a process that can convert L-isoaspartyl residues in peptides to normal L-aspartyl residues. The affinity of a recombinant human methyltransferase for HPr-1, a form deamidated at Asn-38, was relatively poor (Km = 3.6 mM), while a greater affinity was found for HPr-2, a form deamidated at both Asn-12 and Asn-38 (Km = 197 microM). When HPr-2 was incubated with S-adenosylmethionine and the methyltransferase, the bulk of the L-isoaspartyl residues at position 12 was converted to L-aspartyl residues. The major-by-product was the D-isoaspartyl form. The conversion of L-isoaspartyl residues at position 38 to L-aspartyl residues was less complete, reflecting the lower affinity of the methyltransferase for this site. The phosphohydrolysis activity of the repaired form was found to be midway between the form containing only L-aspartyl residues at positions 12 and 38 and the deamidated HPr-2 form.

Structural comparison of the histidine-containing phosphocarrier protein HPr.

The phosphocarrier protein HPr is a central component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) that is responsible for carbohydrate uptake in many bacterial species. A number of three-dimensional structures of HPrs from both Gram-positive and Gram-negative bacteria have been determined; the overall folding topology of HPr is an open-faced beta-sandwich composed of three alpha-helices and a beta-sheet. A detailed structural comparison of these HPrs has been carried out. Besides the overall main chain folding, many detailed structural features are well conserved in all HPr structures. The three x-ray structures of HPrs from Escherichia coli, Streptococcus faecalis, and Bacillus subtilis show considerable overall similarity with respect to the positions of the C alpha atoms. A significant structural difference between HPrs from Gram-positive and Gram-negative bacteria is found in the region of Gly54, owing to the steric effects of Tyr37 in HPrs from the Gram-positive species. The region around Gly54 is involved in the binding of HPr to other PTS proteins and the differences in this region may be responsible for some of the poor functional complementation between HPrs from Gram-positive and Gram-negative species. The active center region, residues 12-18, appears to have significant differences in the comparisons between the overall structures. These differences support the proposal that phosphorylation and dephosphorylation of the active site His15 is accompanied by conformational changes. However, a local structural comparison of residues 12-18 from the x-ray structures of HPrs from E. coli and B. subtilis, and the two-dimensional nuclear magnetic resonance structure of B. subtilis HPr suggests that there is a conserved active center involving residues His15, Arg 17, and Pro18, which shows little conformational change during the phosphorylation cycle. The results of other experimental approaches, including site-directed mutagenesis and NMR spectroscopy, are in some cases difficult to rationalize with some of the details of the structures, but do appear to favour the conclusion that little conformational change occurs.

The 2.0-A resolution structure of Escherichia coli histidine-containing phosphocarrier protein HPr. A redetermination.

The x-ray structure of Escherichia coli HPr has been redetermined at 2.0-A resolution. In contrast to the previous study (El-Kabbani, O. A. L., Waygood, E. B., and Delbaere, L. T. J. (1987) J. Biol. Chem. 262, 12926-12929), the overall structure is, in general, similar to other reported NMR and x-ray HPr structures, although there are some important differences in detail. The overall folding topology of HPr is a classical open-faced beta-sandwich, consisting of four antiparallel beta-strands and three alpha-helices. The least square refinement produced an R index of 0.135 for all measured unique data between 8.0 and 2.0 A resolution. The active center consists of His15 which is hydrogen bonded to a sulfate anion, and Arg17 which has a fully open conformation. This corresponds to the first observed "semi-closed" conformation of the active center of HPr. The Streptococcus faecalis HPr structure (Jia, Z., Vandonselaar, M., Quail, J. W., and Delbaere, L. T. J. (1993) Nature 361, 94-97) has the "open" conformation in which the side chains of His15 and Arg17 are directed as far away from each other as possible. The Bacillus subtilis HPr (Herzberg, O., Reddy, P., Sutrina, S., Saier, M. H., Jr., Reizer, J., and Kapadia, G. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2499-2503) has the "closed" conformation in which the side chains of His15 and Arg17 are close together with a sulfate anion located in the active center. The open conformation represents the unphosphorylated form of HPr whereas the closed conformation likely resembles the phosphorylated form of HPr. The semi-closed conformation observed in the E. coli HPr structure could represent a structural intermediate on the phosphorylation/dephosphorylation pathway of HPr.

Deamidation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system, involves asparagine 38 (HPr-1) and asparagine 12 (HPr-2) in isoaspartyl acid formation.

Histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, when incubated at elevated temperatures forms many species of protein. The two major species are HPr-1 and HPr-2, which have been shown to lack one or two amides, respectively (Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 7023-7033). The formation of HPr-1 and HPr-2 is shown to be pH-dependent and does not occur readily below pH 6. Investigation of the identities and properties of the two residues that deamidate involved creation of site-directed mutants at the 6 glutamine and 2 asparagine residues of HPr; description of their deamidation species by isoelectric focusing; determination of their relative antibody binding properties; assay of their phosphoacceptor and phosphodonor activities; characterization of tryptic and V8-protease peptides; obtaining two-dimensional nuclear magnetic resonance spectra of HPr, HPr-1, and several mutants. It was determined that the sequential deamidation of Asn-38 and Asn-12 yields HPr-1 and HPr-2. Both residues exist as Asn-Gly pairs, and both deamidations probably form isoaspartyl acid. HPr from Bacillus subtilis and Staphylococcus carnosus which also have Asn-Gly at residues 38 and 39 form HPr-1 species presumably by deamidation. HPr from Streptococcus faecalis which does not have Asn-38 does not form a HPr-1 species. The E. coli mutant HPrs, N12D and Q51E, residues that may be involved in the active site, had impaired phosphohydrolysis properties and decreased phosphoenolpyruvate:sugar phosphotransferase system activity.

The involvement of the arginine 17 residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site His-15 that is phosphorylated to form N delta 1-P-histidine. The nearby conserved residue, Arg-17, has been replaced by: lysine, histidine, glutamate, glycine, serine, and cysteine. All mutations resulted in impairment of the phosphoacceptor function of HPr with enzyme I: kcat/Km values between 6% (Ser-17) and 0.1% (Glu-17), relative to wild type. Several sugar-specific enzymes II had different responses. Both the Vmax and Km of enzyme IIN-acetylglucosamine were altered, while for enzyme IImannose only Km was affected, except for R17E. For both enzymes, kcat/Km values were between 0.5 and 3%, with R17E being 10-fold lower. Except for R17E, minimal effects were observed for enzyme IImannitol. These results suggest that there are different rate-limiting steps in the enzymes II. Phosphohydrolysis properties and the pKa values for His-15 and phosphorylated His-15 determined by NMR for both wild type and mutant HPrs suggest that Arg-17 is partly responsible for the instability of P-His-15 and the depressed pKa values in wild type HPr. Other feature(s) of the tertiary structure influence the protonation of His-15 and the phosphohydrolysis properties of phosphorylated His-15.