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J. Biol. Chem., Vol. 275, Issue 42, 33102-33109, October 20, 2000

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The Transport/Phosphorylation of N,N'-Diacetylchitobiose in Escherichia coli

CHARACTERIZATION OF PHOSPHO-IIB^Chb AND OF A POTENTIAL TRANSITION STATE ANALOGUE IN THE PHOSPHOTRANSFER REACTION BETWEEN THE PROTEINS IIA^Chb AND IIB^Chb*

Nemat O. Keyhani, Kirsten Bacia^§, and Saul Roseman^¶

From the Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218

Received for publication, February 8, 2000, and in revised form, May 25, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Enzyme II permeases of the phosphoenolpyruvate:glycose phosphotransferase system comprise one to five separately encoded polypeptides, but most contain similar domains (IIA, IIB, and IIC). The phosphoryl group is transferred from one domain to another, with histidine as the phosphoryl acceptor in IIA and cysteine as the acceptor in certain IIB domains. IIB^Chb is a phosphocarrier in the uptake/phosphorylation of the chitin disaccharide, (GlcNAc)₂ by Escherichia coli and is unusual because it is separately encoded and soluble. Both the crystal and solution structures of a IIB^Chb mutant (C10S) have been reported. In the present studies, homogeneous phospho-IIB^Chb was isolated, and the phosphoryl-Cys linkage was established by ³¹P NMR spectroscopy. Rate constants for the hydrolysis of phospho-IIB^Chb plotted versus pH gave the same shape peak reported for the model compound, butyl thiophosphate, but was shifted about 4 pH units. Evidence is presented for a stable complex between homogeneous Cys10SerIIB^Chb (which cannot be phosphorylated) and phospho-IIA^Chb, but not with IIA^Chb. The complex (a tetramer (3)) contains equimolar quantities of the two proteins and has been chemically cross-linked. It appears to be an analogue of the transition state complex in the reaction: phospho-IIA^Chb + IIB^Chb  IIA^Chb + phospho-IIB^Chb. This is apparently the first report of the isolation of a transition state analogue in a protein-protein phosphotransfer reaction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The accompanying papers¹ present evidence that the chitin disaccharide (GlcNAc)₂² is taken up in Escherichia coli by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The three genes involved in this process were previously characterized as part of a cryptic cellobiose operon (6), and the three proteins were designated IIA^Cel, IIB^Cel, and IIC^Cel, respectively. We suggested (7) that the appropriate nomenclature is IIA^Chb, IIB^Chb, and IIC^Chb (Chb for N-acetylchitobiose). We report here the isolation and characterization of phospho-IIB^Chb. Our concept of how (GlcNAc)₂ is taken up by E. coli is summarized schematically in Fig. 1.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Schematic of the PTS reaction sequence. PEP donates its phosphoryl group to Enzyme I (EI) and then through the chain of proteins, HPr to IIAChb to IIBChb to the sugar that is simultaneously translocated and phosphorylated by the membrane sugar receptor, IICChb. The phosphoryl group is linked to His in Enzyme I, HPr and IIAChb, and to Cys in IIBChb. Enzyme I and IIAChb are homodimers.

In most phospho-PTS proteins, the phosphoryl group is linked to a His residue. However, the active site amino acid in the IIB domain of the E. coli mannitol Enzyme II complex was shown to be cysteine (8), and shortly thereafter the same result was found with the IIB domain of the glucose-specific Enzyme II complex of E. coli (9). The definitive method for characterizing this novel linkage was by ³¹P NMR spectroscopy (see "Discussion"). Sequence similarity of the amino acids around the active site in other IIB domains (10), including IIB^Chb, suggests that the phosphoryl group may be linked to Cys in these proteins as well, although they have not been definitively characterized. In the present studies, homogeneous phospho-IIB^Chb was isolated and the phosphoryl linkage to Cys¹⁰ (the only Cys in the protein) was established by ³¹P NMR.

A C10S mutant of IIB^Chb (or IIB^Cel) has been crystallized, and both its crystal and solution structures have been determined (11, 12). Apparently the Ser replacement was used because the Cys caused technical difficulties. As shown here, the mutant protein cannot be phosphorylated. But perhaps the most significant result reported in the present studies is that Cys10SerIIB^Chb forms a stable complex with phospho-IIA^Chb (but not with IIA^Chb). Further, the complex can be chemically cross-linked. It seems likely that the complex is a transition state analogue for the phosphotransfer reaction between the two native proteins. To our knowledge, there are no reports of the isolation of a transition state complex, or of an analogue of such a complex, involving a protein-protein phosphotransfer reaction.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials and Methods

The materials and biochemical and molecular biological methods are the same as described in the accompanying paper on IIA^Chb (2).

Construction of IIB^Chb Overexpression Vector

The open reading frame corresponding to the chbB gene was cloned into the pET21a (Novagen, Madison, WI) overexpression vector using polymerase chain reaction and primers specific to the ends of the gene. The primers were designed with unique restriction sites at each end to facilitate the cloning procedure. Polymerase chain reaction generated fragments were agarose gel purified and cloned into the pNoTA shuttle vector (5 Prime  3 Prime, Inc., Boulder, CO) and then subsequently subcloned into pET21a using standard procedures. The nucleotide sequences of the primers are given below. The engineered restriction sites are underlined, and the start site of the gene is in bold type. A mutant version of the protein was also constructed in which the active site Cys (amino acid 10) was converted to a Ser. This was achieved by designing a primer with a mismatch in the sequence converting the codon from Cys to Ser, with the mutation given below in underlined italics.

The primers used were: for chbB, 5'-GTCATATGGAAAAGAAACACATTTATCTGT-3' (NdeI) and 5'-GTATTGATTTATGAATTCACTCTTTGACGG-3' (EcoRI); for Cys10SerchbB, 5'-GTCATATGGAAAAGAAACACATTTATCTGTTTTCTTC-3' (NdeI, mutation at amino acid residue 10 results from the change of a G in position 34 of the primer to a C) and the same primer as the second one listed above for chbB. The isolated subclones in pET21a were confirmed by sequencing the entire insert.

Purification of IIB^Chb

The IIB^Chb proteins were purified essentially as described (12). Two liters each of LB media supplemented with 100 µg/ml ampicillin in three 6-liter flasks were inoculated with 40 ml (each) of an overnight culture of E. coli strain BL21 (DE3)EI (containing a deletion in Enzyme I of the PTS) harboring the plasmid pET:chbB (or pET:Cys10SerchbB). The culture was shaken vigorously at 37 °C until A₆₀₀ = 0.8-1.0 (2-3 h) before being induced by the addition of 1 mM (final concentration) isopropyl-1-thio--D-galactopyranoside. Cells were allowed to grow for an additional 2-3 h and harvested by centrifugation at 4000 × g for 10 min at 4 °C. The following steps were conducted at 0-4 °C unless otherwise stated. The cell pellet was washed twice with TG buffer (10 mM Tris, acetate buffer, pH 6.5, containing 1% glycerol, 1 mM NaN₃, and 1 mM DTT) and resuspended in the same buffer using 4.0 ml/g (wet weight) of cells. After passage twice through a French Press, cell debris was removed by centrifugation at 12,000 × g for 15 min. Membranes were removed by high speed centrifugation at 160,000 × g for 1 h.

Step 1: Mono-S-Sepharose Chromatography-- The high speed supernatant (40-50 ml, 5-10 mg protein/ml) was applied to a 2.6 × 15 cm (75 ml) Mono-S-Sepharose column equilibrated in TG buffer, at a flow rate of 1 ml/min, after which the column was washed overnight with TG buffer before being eluted with a 1-liter gradient of 0-300 mM NaCl in TG buffer. Fractions were analyzed by SDS-PAGE. The protein eluted between 100 and 150 mM NaCl. Pooled fractions were concentrated to 5-10 ml (10-20 mg protein/ml) using Centriprep-3 centrifugal filter devices (Millipore).

Step 2: Sephadex G-50 Gel Filtration Chromatography-- A Sephadex G-50 column (2.6 × 82 cm) was equilibrated with TG buffer containing 100 mM NaCl. The pooled, concentrated fractions from Step 1 were transferred to the column and eluted with the same buffer. Protein fractions were pooled based on purity as determined by SDS-PAGE. The purified protein was concentrated as described above (to 5-10 mg/ml) and dialyzed against 25 mM sodium phosphate buffer, pH 8.0. Wild type protein was dialyzed against the same buffer containing 0.2 mM DTT. Purified protein aliquots were stored at 70 °C until used.

Phosphorylation Assay

The assay was performed as described (2) for the phosphorylation of IIA^Chb. For measurement of IIB^Chb phosphorylation, the assay reaction mixture contained (20 µl) 50 mM Tris-HCl buffer, pH 8.0, 10 mM MgCl₂, 1 mM DTT, 5 mM NaF, 2-5 pmol of purified Enzyme I, 5-10 pmol of purified HPr, 2-10 pmol of purified IIA^Chb, and 100-1000 pmol of purified IIB^Chb. Reactions were initiated by the addition of 0.2-2 nmol of [³²P]PEP (10-20 cpm/pmol). Aliquots were taken over the time course, and the reaction was stopped by dilution with 1.0 ml of ice-cold buffer (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl) and filtered through polyvinylidene difluoride filters (Sartorius). The filters were washed twice with 1 ml of the same buffer, immersed in 4 ml of Packard Ultima-Gold XR liquid scintillation counter mixture, and counted in a Packard Liquid Scintillation Spectrometer. Control incubation mixtures lacked either Enzyme I, HPr, or IIA^Chb.

Isolation of Phospho-IIB^Chb

Phosphorylation reactions were carried out in a buffer containing 25 mM sodium phosphate buffer, pH 8.0, 5 mM MgCl₂, and 0.5 mM DTT. Amounts of purified IIB^Chb ranging from 50 nmol to 3.0 µmol were phosphorylated with Enzyme I, HPr, and IIA^Chb at molar ratios from 1:100 to 1:200 relative to the IIB^Chb. Reactions were initiated by adding a 30-fold molar excess (with respect to IIB^Chb) of PEP and incubated at 37 °C for 45 min, after which an additional 10-fold molar excess of PEP was added and the reaction was allowed to continue for 30 min. The phospho-IIB^Chb was purified either by native gel electrophoresis and electroelution of the appropriate segment of the gel or by purification on a Superdex-75 gel filtration column (fast protein liquid chromatography) as described below.

Kinetics of Hydrolysis of Phosphoprotein as a Function of pH

The rate of hydrolysis of [³²P]phospho-IIB^Chb was determined as a function of pH at 25 °C. The following buffers were used: McIlvaine's sodium phosphate-citric acid broad range buffer from pH 2.0 to 8.8, Bates and Bowers boric acid-KCl buffer (pH 8.0-10), sodium phosphate, sodium acetate, sodium borate, Tris-HCl, MOPS-HCl, and TAPS-HCl. At each pH, the kinetics of hydrolysis were determined using the DEAE-paper method for separating [³²P]P_i from [³²P]phospho-IIB^Chb (13), and initial rates were determined to calculate the respective rate constants.

Detection, Isolation, and Analysis of a Complex between Phospho-IIA^Chb and Cys10SerIIB^Chb

The mutant IIB^Chb protein could not be phosphorylated, as expected. However, when stoichiometric amounts of phospho-IIA^Chb were added to the mutant protein, a complex was detected using native gel electrophoresis (see "Results"). Two methods were developed to purify the complex.

Electroelution from Native Gel-- Equimolar amounts of IIA^Chb and IIB^Chb were incubated with Enzyme I and HPr at 1:250 the molar concentrations of IIA^Chb and IIB^Chb. Reactions were initiated by adding a 40-fold excess of PEP and allowed to incubate for 1 h at 37 °C. Samples were electrophoresed in a 16% polyacrylamide gels under native conditions, and the protein band corresponding to the complex was electroeleuted from the gel.

Superdex-75 Gel Filtration-- A two-step procedure was employed to purify the complex. First, IIA^Chb was phosphorylated and purified by gel filtration chromatography using a Superdex-75 (10 × 300 mm; Amersham Pharmacia Biotech) fast protein liquid chromatography column and system. The column was equilibrated and eluted with 25 mM sodium phosphate pH 8.0 buffer. The protein concentration in the pooled fractions containing the purifed phospho-IIA^Chb was estimated by A₂₈₀ and a 1.1-fold excess Cys10SerIIB^Chb was added to the phosphoprotein. The mixture was maintained at room temperature for 15-20 min before being transferred to a Superdex-75 gel filtration column. The three proteins (IIB^Chb, IIA^Chb, and the complex) could easily be separated on the column (see "Results").

Cross-linking Experiments

Phosphorylation reactions were performed in 25 mM sodium phosphate, pH 8.0, buffer containing 5 mM MgCl₂ and 0.2 mM DTT. Typical reaction mixtures (10 µl) contained equimolar amounts (0.7-1.4 nmol) of IIA^Chb and Cys10SerIIB^Chb, 5-10 pmol of Enzyme I, 5-10 pmol of HPr, and 20-50 nmol of PEP. Reaction mixtures were incubated at 37 °C for 30 min prior to the addition of the cross-linking reagent (20-30-fold excess) and then allowed to stand at room temperature for 30-60 min. Unless otherwise indicated, reactions were quenched by adding 1 µl of 0.5 M Tris-HCl buffer, pH 7.5, and incubated for an additional 5 min at room temperature. SDS loading buffer (5 µl) was then added, and samples were analyzed by SDS-PAGE. The following cross-linking reagents (Pierce) were tested: bis(sulfosuccinimidyl)suberate (BS³), 1-ethyl-3-(dimethylaminopropyl)carbodiimide, dimethyladipimidate, dimethylsuberimidate, disuccinimidyl tartrate, dithiobis(succinimidyl propionate), and 3,3'-dithiobis(sulfosuccinimidyl proprionate) (DTSSP). Stock solutions (10-25 mM) of BS³, dimethyladipimidate, dimethylsuberimidate, and DTSSP were prepared in 25 mM sodium phosphate buffer, pH 8.0, and solutions of disuccinimidyl tartrate and dithiobis(succinimidyl propionate) were prepared in Me₂SO. For reactions containing the cross-linkers dithiobis(succinimidyl propionate) and DTSSP, DTT was omitted from all buffers and solutions. 1-Ethyl-3-(dimethylaminopropyl)carbodiimide reaction mixtures were quenched using 2-mercaptoethanol (final concentration, 10 mM) instead of Tris-HCl buffer.

Analysis of DTSSP Cross-linked Product

A 10-fold scaled up cross-linking reaction was performed as described above using DTSSP as the cross-linking reagent. The reaction mixture (100 µl) contained 12 nmol of IIA^Chb and Cys10SerIIB^Chb, 50 pmol of Enzyme I, 50 pmol of HPr, and 50 nmol of PEP in buffer (25 mM sodium phosphate buffer, pH 8.0, containing 5 mM MgCl₂). The sample was analyzed by SDS-PAGE, and the band corresponding to a 26-kDa molecular mass protein was electroeluted from the gel. After electroelution a portion of the sample (100 µl) was treated with DTT (10 mM) for 30 min and then dialyzed against (100 ml) 25 mM sodium phosphate buffer, pH 8.0, prior to SDS-PAGE analysis.

³¹P NMR Spectroscopy

The NMR experiments were kindly performed by Dr. Charles Long (Department of Chemistry, Johns Hopkins University). A large scale sample (3.0 µmol) of IIB^Chb was phosphorylated and purified as described above. The final purified sample was concentrated to 1.0 ml (2.5 mM, 83% yield) and exchanged with 80% D₂O. The sample was transferred to a 5-mm NMR tube, and a 5-mm broad band ³¹P probe was used for all measurements. NMR spectra were recorded on a 500-MHz Varian Unity Plus spectrometer operating at 202 Mhz (³¹P frequency), using a 10-µs (53°) pulse and a repetition time of 3.2 s. Protons were decoupled by broad band decoupling. All spectra were recorded at 10 °C, pH 8.2-8.8 (calculated to be pH 8.6). Chemical shifts are reported relative to an external standard of 85% phosphoric acid, which was set to 0.0 ppm, and repeatedly checked for drift.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of IIB^Chb-- IIB^Chb was overexpressed and purified from E. coli BL21:EI harboring pET:IIB^Chb. As in the case of IIA^Chb (2), IIB^Chb was purified from a deletion of Enzyme I to ensure that it was isolated in its unphosphorylated form.

SDS-PAGE of the purified protein is shown in Fig. 2A. The protein migrates with an apparent molecular mass of 11-12 kDa, which agrees with a predicated molecular mass of 11,400 Da from the gene sequence (see Ref. 7). The protein is not processed during expression because the N-terminal amino acid sequence agreed with that predicted from the coding sequence (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Purity and phosphorylation of recombinant IIBChb. A, SDS-PAGE. IIBChb and Cys10SerIIBChb were purified as described under "Experimental Procedures." The proteins (5 µg each) were analyzed by SDS-PAGE (16% polyacrylamide gel) and stained with Coomassie Brilliant Blue. Lane 1, molecular mass standards; lane 2, wild type IIBChb; lane 3, Cys10SerIIBChb. B, phosphorylation of IIBChb. Phosphorylation was assayed by native gel electrophoresis (no SDS, 16% polyacrylamide gel). Reaction mixtures (20 µl) were incubated at 37 °C for 1 h and contained 25 mM sodium phosphate buffer, pH 8.0, 5 mM MgCl2, 0.1 µg of Enzyme I, and 0.1 µg of HPr. Lane 1, IIAChb (4 µg) without PEP; lane 2, IIAChb (4 µg) + 10 mM PEP; lane 3, IIBChb (4 µg); lane 4, IIAChb (0.1 µg) + IIBChb (4 µg) + 10 mM PEP; lane 5, IIAChb (4 µg) + IIBChb (4 µg) + 10 mM PEP. After incubation, 5 µl of native gel loading buffer (125 mM Tris base, 1.0 M glycine, 0.1 M DTT, 25% glycerol, and 0.005% bromphenol blue) was added to each reaction tube before samples were applied and electrophoresed (16% polyacrylamide) under navtive conditions. The gel was stained using Coomassie Brilliant Blue. Phospho-IIAChb and phospho-IIBChb were also visualized in reaction mixtures containing [32P]PEP by autoradiography (not shown). C, ki- netics and requirements for phosphorylation of IIBChb. Phosphorylation was measured by DEAE-paper chromatography as described under "Experimental Procedures." Aliquots (50 µl) were taken over the indicated time course from 0.5-ml reaction mixtures incubated at 37 °C. Each reaction mixture contained 25 mM sodium phosphate buffer, pH 8.0, 5 mM MgCl2, 5 mM [32P]PEP and the following proteins: , IIBChb (450 pmol), Enzyme I (4.5 pmol), HPr (4.5 pmol), IIAChb, (4.5 pmol); , IIBChb (450 pmol), Enzyme I (2.25 pmol), HPr (2.25 pmol), IIAChb (2.25 pmol). Controls () were incubation mixtures where: (a) Enzyme I, HPr, IIAChb, or PEP were omitted; (b) IIAGlc was substituted for IIAChb; and (c) Cys10SerIIBChb was substituted for IIBChb.

Phosphorylation of IIB^Chb-- Although SDS-PAGE cannot separate IIB^Chb and phospho-IIB^Chb, the proteins are separable by native gel electrophoresis (Fig. 2B). Densitometric scans of the stained gels permitted quantitation of the two proteins and were used to determine the extent of phosphorylation of IIB^Chb.

The kinetics of phosphorylation of IIB^Chb are shown in Fig. 2C. IIB^Chb was incubated with PEP, Mg²⁺, and catalytic quantities of homogeneous Enzyme I, HPr, and IIA^Chb. No phospho-IIB^Chb was detected when any of the four proteins was omitted from the incubation. Thus, there is no detectable transfer from phospho-HPr to IIB^Chb. Direct transfer of the phosphoryl group from phospho-IIA^Chb to IIB^Chb was also demonstrated (data not shown), and the kinetics of this reaction will be presented elsewhere.

The phosphoprotein was isolated in mg quantities, and after gel chromatography for final purification and analysis by native gel electrophoresis, it was used for the following studies. Preparations of phospho-IIB^Chb were used only when there was no detectable unphosphorylated protein (less than 5%).

³¹P NMR Spectrum of Phospho-IIB^Chb-- ³¹P NMR spectroscopy is a powerful tool for determining the structures of phosphorylated compounds. For example, there is a significant and specific chemical shift in for virtually each phosphoryl linkage found in proteins (for reviews see Refs. 14 and 15).

The ³¹P NMR spectrum of homogenous phospho-IIB^Chb is shown in Fig. 3 relative to 85% phosphoric acid. A single signal was observed downfield, at 16.3 ppm at 10 °C, pH 8.6. 

View larger version (21K): [in this window] [in a new window]   Fig. 3.   31P NMR of phospho-IIBChb. 31P NMR spectra of purified phospho-IIBChb were recorded as described under "Experimental Procedures." A, spectral accumulations at 81 min, 3 Hz line broadening, 1536 transients collected, 9728 data points. B, 17 h, 10 Hz line broadening, 19,200 transients collected, 9728 data points.

This 16.3 ppm signal can be compared with those reported for phosphocysteine in the following: (a) The IIB domain of the E. coli Enzyme II^Mtl (16), + 11.9 ppm at pH 8.0 relative to P_i, or approximately +14-15 relative to 85% H₃PO₄. The model compound, synthetic phosphocysteamine gave a signal at +13.3 ppm and, when corrected relative to 85% H₃PO₄, was at +15.3-16.3 ppm. (b) The IIB domain of the E. coli Enzyme II^Glc (17), +11.7 ppm at pH 6.8 relative to P_i or ~13.7-15 relative to 85% H₃PO₄. (c) The IIB domain of the Staphylococcus carnosus Enzyme II^Mtl (18) was cloned linked to a His tag (for purification), +13.8 ppm at 10 °C, pH 7.5 relative to 85% H₃PO₄. (d) Both a synthetic phosphocysteinylpeptide and a leukocyte tyrosine phosphatase phosphoprotein intermediate (19) in the hydrolytic reaction showed a chemical shift of +16.1 ppm in the rapid quench fluid (0.2 N NaOH) relative to 85% H₃PO₄, where the P_i signal was +5.5 ppm. (e) A similar human dual specific phosphatase (20) showed a chemical shift at +13.7 ppm relative to 85% H₃PO₄ at pH 7.0, 22 °C.

The chemical shifts observed with thiophosphoryl derivatives are far downfield from all other known ³¹P chemical shifts for both low molecular mass phosphoryl derivatives and phosphoproteins (14, 15). These include O-phosphoserine and threonine, N-phospholysine, N-phosphoarginine, O-phosphotyrosine, acylphosphate (e.g. to aspartate), phospho-N²- and phospho-N¹-histidine, and the pyrophosphate linkage. At pH 8.0, the chemical shifts for these substances lie in the range + 4 to 11 relative to 85% H₃PO₄ set at 0 ppm. Our ³¹P NMR results therefore lead to the conclusion that phosphoryl group in phospho-IIB^Chb is linked to Cys¹⁰. This linkage had previously been surmised based on amino acid sequence similarity but had not been experimentally demonstrated.

The first spectrum shown in Fig. 3 was collected over a period of 81 min at pH 8.6, 10 °C. On repeated scans of the sample, over a period of 17 h, another peak was observed at +3.1 ppm, which increased with time, whereas the peak at +16.3 ppm decreased. The new peak was identified as inorganic phosphate, corresponding in its chemical shift to a standard in the same buffer (data not shown). Phospho-IIB^Chb therefore slowly hydrolyzes under these conditions.

Stability of Phospho-IIB^Chb-- Aside from the early studies on the properties of a thiophosphate ester, butyl thiophosphate (21), there are only a few reports on the properties of the thiophosphate group in proteins or peptides derived by proteolysis of the phosphoprotein.

The rate constants for the hydrolysis of [³²P]phospho-IIB^Chb as a function of pH at 25 °C are shown in Fig. 4. The maximum instability is pH ~8. At this pH, phospho-IIB^Chb is hydrolyzed at about seven times the rate of phospho-IIA^Chb at the concentration used for the latter (2).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of pH on the rate of hydrolysis of phospho-IIBChb and on the complex (phospho-IIAChb:Cys10SerIIBChb). 32P-Labeled phospho-protein or complex was purified by electroelution from a native gel or by gel filtration on a Superdex-75 fast protein liquid chromatography column as described under "Experimental Procedures." Assays for hydrolysis were conducted at 25 °C by the DEAE-paper chromatography method. Rate constants were determined from logarithmic plots (phosphoprotein remaining versus time) as a function of pH in the following buffers: pH 2-8, McIlvaine's: pH 8-10, Bates and Bowers. Other buffers (see "Experimental Procedures") were also tested, and gave essentially the same results: , phospho-IIBChb; , the complex formed by phospho-IIAChb and Cys10SerIIBChb. The dashed line represents data for the rate of hydrolysis of phospho-IIAChb taken from the accompanying paper (2).

The bell-shaped curve in Fig. 4 is similar in shape to that observed for the spontaneous hydrolysis of the model compound, butyl thiophosphate (21), except that the peak of the curve for the latter has a pH of ~3-4. The shift to the right of about 4 pH units for the phosphoprotein must reflect the effects of the local environment surrounding the thiophosphoryl group in the protein. Different results were obtained with other thiophosphates: (a) E. coli phospho-Enzyme II^Mtl was subjected to trypsin and chymotrypsin digestion (8), a phosphopeptide (14 amino acids) was isolated, and the phosphoryl group was found to be linked to Cys (the first such report). The stability of the phosphopeptide was studied in the range pH 2-13 and gave an inverted bell-shaped curve, with maximum instability at pH 2-4 and 13 and maximum stability at pH 10-12. (b) Similar results were obtained with a phosphododecapeptide isolated from the E. coli IIBC^Glc transporter (22). The curve, resembling an hyperbola, was virtually superimposable over that obtained with the II^Mtl phosphopeptide in the range pH 2-12. (c) Finally, the intact phosphoprotein-tyrosine phosphatase showed a bell-shaped profile, similar to the model compound butyl thiophosphate, with maximum instability at pH 2-3 and maximum stability at pH 8-10 (23). Thus, phospho-IIB^Chb behaves differently from other known thiophosphates with respect to its stability as a function of pH. This observation is discussed below.

Formation of Complex between Phospho-IIA^Chb and Cys10SerIIB^Chb-- When [³²P]phospho-IIA^Chb and the mutant protein Cys10SerIIB^Chb were mixed in equimolar quantities and subjected to native gel electrophoresis, a new labeled band was observed in the gel (Fig. 5A), intermediate in its position between the two proteins. The band was cut from the gel, eluted, and analyzed by SDS-PAGE, and [³²P]phospho-IIA^Chb and Cys10SerIIB^Chb were found (Fig. 5B). The two proteins were present in equimolar quantities in the complex, as indicated by quantitative densitometry of the bands (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Complex formation between phospho-IIAChb and Cys10SerIIBChb (native gel electrophoresis). A, reaction mixtures (20 µl) were incubated at 37 °C for 1 h and contained 25 mM sodium phosphate buffer, pH 8.0, 5 mM MgCl2, 0.1 µg of Enzyme I, 0.1 µg of HPr, and the following components: lane 1, IIAChb (4 µg); lane 2, IIAChb (4 µg) + 10 mM PEP; lane 3, Cys10SerIIBChb (4 µg) + 10 mM PEP; lane 4, IIAChb (0.08 µg) + Cys10SerIIBChb (4 µg) + 10 mM PEP; lane 5, IIAChb (4 µg) + Cys10SerIIBChb (4 µg) + 10 mM PEP. After incubation, 5 µl of native gel loading buffer (125 mM Tris base, 1.0 M glycine, 0.1 M DTT, 25% glycerol, and 0.005% bromphenol blue) was added to each reaction tube before samples were applied and electrophoresed (16% polyacrylamide) under native conditions. The gel was stained with Coomassie Brilliant Blue. Phospho-IIAChb and the complex was also visualized in reaction mixtures containing [32P]PEP by autoradiography (not shown). B, SDS-PAGE of purified complex. The band indicated as the complex in A was purified either by electroelution from a native gel (lane 1) or by gel filtration chromatography (lane 2) as described (Fig. 6) and then analyzed by SDS-PAGE (denaturing conditions, 16% polyacrylamide gel). Phospho-IIAChb and the complex was also visualized in reaction mixtures containing [32P]PEP by autoradiography (not shown).

The complex was also isolated by gel filtration chromatography (Fig. 6). In this experiment, [³²P]phospho-IIA^Chb was added to a 10% excess of Cys10SerIIB^Chb, and the mixture was fractionated by gel filtration chromatography using a Superdex-75 column. The first and largest protein peak contained the ³²P followed by a small peak of Cys10SerIIB^Chb. Fig. 6 shows that the higher molecular mass or major peak (labeled Complex) is eluted before the standards used to calibrate the column, including IIA^Chb or phospho-IIA^Chb, which are not separated by this method. No complex was detected with any of the following combinations of IIA and IIB proteins: (IIA^Chb or phospho- IIA^Chb) plus (IIB^Chb or phospho-IIB^Chb); IIA^Chb and Cys10SerIIB^Chb. The higher molecular mass radiolabeled peak from the column in Fig. 6 was analyzed by SDS-PAGE and, as shown in Fig. 5B, contained both of the starting proteins, [³²P]phospho-IIA^Chb and Cys10SerIIB^Chb, in equimolar quantities.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Separation of IIAChb, IIBChb, and of the complex using Superdex-75 gel chromatography. A Superdex-75 FLPC column and chromatography system (Amersham Pharmacia Biotech) was equilibrated in 25 mM sodium phosphate buffer, pH 8.0, and after loading the protein solutions (0.5 ml each), was eluted with the same buffer at a flow rate of 0.5 ml/min. The results of three experiments are shown and labeled as follows. Complex (solid line), 75 nmol of phospho-IIAChb and 85 nmol Cys10SerIIBChb were mixed, incubated 1 h at 37 °C, and transferred to the column. IIAChb (dotted line), 2 mg of IIAChb or phospho-IIAChb. IIBChb (dashed line), 0.5-0.75 mg of Cys10SerIIBChb, IIBChb, or phospho-IIBChb.

From these results we conclude that phospho-IIA^Chb and Cys10SerIIB^Chb form a stable complex containing equimolar quantities of the proteins and that IIA^Chb does not substitute for phospho-IIA^Chb. The binding constant holding the proteins in the complex is sufficiently high so that they do not separate on a gel filtration column. The same results were obtained by subjecting the complex eluted from the column to analytical ultracentrifugation (3). A schematic version of these and the following results is shown in Fig. 7.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Schematic of complex (cross-linking). The cartoon depicts the reactions leading to the cross-linked complex. Admixture of 2 mol of the Cys10SerIIBChb mutant protein and 1 mol of phospho-IIAChb dimer yields a complex that is stable to electrophoresis and to Sephadex gel filtration chromatography but unstable under conditions used in attempts to crystallize it. The complex was therefore treated with BS3, which yielded a cross-linked product. Under denaturing conditions (SDS-PAGE), the noncovalent bonds that link the phospho-IIA monomers in the heterotetramer are dissociated to yield 2 mols of heterodimer.

The stability of the phosphoryl linkage in the ³²P-labeled complex was studied as a function of pH, over the range 6-11 (Fig. 4). The complex precipitates at lower pH values. Unexpectedly, we found that the complex was slightly less stable than phospho-IIA^Chb, suggesting that the phosphoryl linkage is not shielded from attack by the solvent in the complex.

Cross-linking of Phospho-IIA^Chb and Cys10SerIIB^Chb-- When efforts were made to crystallize the complex, it partially dissociated. To eliminate this problem, attempts were made to covalently cross-link the two proteins in the complex. A number of reagents were tested, and the most satisfactory results were obtained with BS³, a noncleavable cross-linking reagent that reacts with amines. When the reaction products were subjected to SDS-PAGE, they gave the results shown in Fig. 8. A major band was detected in the gel that migrated at a molecular mass of 24-25 kDa. The calculated molecular masses (not corrected for the cross-linkers) of potential products of a reaction mixture containing phospho-IIA^Chb and Cys10SerIIB^Chb are: the homodimer (phospho-IIA)₂, 25 kDa; the homodimer (IIB)₂, 22.8 kDa; the heterodimer phospho-IIA/IIB, 24.3; and the heterotetramer (phospho-IIA/IIB)₂, 48.3. The observed band could be either of the two homodimers or the desired product, a heterodimer of the two proteins, which may have been derived from the tetramer upon SDS treatment (Fig. 7).

View larger version (79K): [in this window] [in a new window]   Fig. 8.   Analysis of cross-linked products of IIAChb and Cys10SerIIBChb using BS3. The cross-linked products of IIAChb and IIBChb under a variety of conditions was monitored by SDS-PAGE (16% polyacrylamide gels). Reactions were performed as described under "Experimental Procedures." All reaction were performed in 25 mM sodium phosphate buffer, pH 8.0, containing 0.1 mM DTT, 5 mM MgCl2, and 10 mM PEP. Where indicated in the table at the bottom, the mixtures were treated with 0.6 mM BS3. The following protein concentrations were used per reaction mixture (20 µl): 0.2 µg of Enzyme I, 0.2 µg of HPr, 5 µg of IIAChb, and 5 µg of Cys10SerIIBChb. Reactions were quenched and mixed with 5 µl of gel loading dye. The samples were boiled for 3 min before transferring to the gel.

Control experiments offered strong evidence that the 24-25-kDa band consisted of the desired product. Fig. 8 shows that the new protein band was formed when the reaction mixture contained phospho-IIA^Chb and the Cys10SerIIB^Chb (lane 3). A number of other minor protein bands are evident in lane 3 that appear to arise from the reaction of IIA^Chb or of phospho-IIA^Chb with the cross-linking reagent (lanes 4 and 7, respectively). By contrast, both IIB^Chb and phospho-IIB^Chb showed little reactivity with the cross-linking reagent (lanes 5 and 9, respectively). (The phosphorylating system proteins Enzyme I and HPr were used at such low concentrations that they showed no cross-linked products on the gel (lane 8)). There may have been a small amount of cross-linked complex formed between dephospho IIA^Chb and the Cys10SerIIB^Chb mutant protein (lane 6), because a new band appears that migrates at about the same rate as the major protein band (the putative complex) in lane 3. It is, of course, possible that IIA^Chb and the Cys10SerIIB^Chb associate to form a small quantity of a complex, but that this quantity increases because the cross-linker shifts the equilibrium toward the formation of more complex. Finally, it was surprising to find that neither IIA^Chb nor phospho-IIA^Chb, which form such tight dimers, yield any significant quantity of cross-linked dimers under these conditions (lanes 4 and 7, respectively), although higher molecular mass products were generated.

To further establish the composition of the 24-25-kDa band (lane 3), the two proteins were subjected to a cross-linking reaction with DTSSP, a cleavable disulfide analogue of BS³. The cleavable disulfide linkage in DTSSP replaces two methylene groups in the suberate chain of BS³; the spacer arm is 11.4 Da in BS³ and 12D in DTSSP. The DTSSP reaction product migrated at 24-25 kDa in the SDS gel. After elution from the gel, the product was treated with DTT, and the reaction mixture was subjected again to SDS-PAGE. Two protein bands were observed, corresponding to the reactants, phospho-IIA^Chb and Cys10SerIIB^Chb (Fig. 9). The results of both sets of experiments therefore indicate that the cross-linked product obtained with BS³ is the desired complex, containing phospho-IIA^Chb and Cys10SerIIB^Chb.

View larger version (65K): [in this window] [in a new window]   Fig. 9.   Analysis of purified DTSSP cross-linked product. A cross-linking reaction was performed as described under "Experimental Procedures" using DTSSP. The cross-linked product was purified by electroelution from SDS-PAGE (16% polyacrylamide gel). The purified product was reanalyzed by SDS-PAGE (16% polyacrylamide gel). Lane 1, molecular mass standards; lane 2, 8 µg of purified product treated with 10 mM DTT for 30 min and dialyzed as described, prior to electrophoresis; lane 3, 5 µg of purified cross-linked product not treated with DTT.

The analyses were performed with SDS-PAGE. Because the native dimer (phospho-IIA^Chb)₂ is dissociated to its monomers under these conditions, the covalently cross-linked complex could be the heterodimer phospho-IIA/IIB or the heterotetramer (phospho-IIA/IIB)₂ (Fig. 7). Both the gel filtration and analytical ultracentrifugation experiments suggest that the native cross-linked complex is, in fact, the tetramer, schematically illustrated in Fig. 7.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although the solution and crystal structures of the mutant protein Cys10SerIIB^Chb have been established (11, 12) and IIB^Chb has been isolated, there are no reports on the properties of phospho-IIB^Chb. As demonstrated by analytical sedimentation (3), IIB^Chb and phospho-IIB^Chb are monomeric, unlike both IIA^Chb and phospho-IIA^Chb, which form stable dimers. In addition, we show that phosphate transfer proceeds as depicted in Fig. 1, from PEP through Enzyme I, HPr to IIA^Chb to IIB^Chb, and finally to (GlcNAc)₂, the last step mediated by the membrane receptor, IIC^Chb. Kinetic and thermodynamic studies on the reversible transfer of the phosphoryl group between IIA^Chb and IIB^Chb are now in progress.

Early work on phospho-PTS proteins established that the phosphoryl group was generally linked to a histidine. However, in 1988 Pas and Robillard (8, 24) reported a unique linkage, a thiophosphate, in a phosphoryl-polypeptide isolated from the IIB domain of II^Mtl. The thiophosphoryl linkage was subsequently reported in the IIB domain of IICB^Glc (22) and in the IIB domain of the Staphylococcus carnosus Enzyme II^Mtl (18). Amino acid sequence similarities also suggested that Cys is the active site amino acid in a number of IIB domains. The thiophosphoryl linkage has also been found in a different family of enzymes, protein-tyrosine phosphatases (23), where they act as catalytic intermediates in the overall hydrolysis reaction (19, 20).

The phosphoryl group in phospho-IIB^Chb was presumed to be linked to Cys¹⁰ (25) based on sequence similarity. In the present studies, phospho-IIB^Chb was isolated in homogeneous form, and the linkage was shown to be a thiophosphate by ³¹P NMR; IIB^Chb contains only one Cys. IIB^Chb offers a unique advantage for these experiments because it is a separately encoded protein. Thus, NMR can be directly applied without resorting to protease digestion and the isolation of a phosphopeptide and therefore without the danger of an artifactual result because of phosphoryl migration.

For structural and other experiments on phospho-IIB^Chb, it was necessary to determine its stability. Like butyl thiophosphate (21), the curve of hydrolysis rate versus pH is bell-shaped, but whereas butyl thiophosphate shows maximum instability at pH ~3-4, phospho-IIB^Chb shows maximum instability at pH ~8. This behavior is different from thiophosphopeptides isolated from other IIB domains and from that of a protein-tyrosine phosphatase, all of which more closely resemble butyl thiophosphate. The most likely explanation for this large difference in behavior of the thiophosphate group in phospho-IIB^Chb are neighboring group effects. It should be noted that IIB^Chb is a very basic protein, with a calculated pI of 8.0. Furthermore, two crystal structures have been solved, one a protein-tyrosine phosphate phosphatase mutant with the thiophosphoryl group intact (26), and the second the corresponding native enzyme from Yersinia liganded to tungstate (in place of covalent phosphate) (27). In both cases, the structures show that a conformationally flexible loop closes over the phosphate (or tungstate), thereby increasing the number of amino acid side chains surrounding the phosphoryl moiety. The ³¹P NMR results obtained here with phospho-IIB^Chb are also consistent with the idea of strong neighboring groups interactions, because the 16.3 ppm downfield shift at pH 8.6 was significantly greater than in the other thiophosphoryl proteins and was similar to that of the leukocyte tyrosine phosphatase phosphoprotein, where the chemical shift was +16.1 in 0.2 N NaOH (19). In other words, the phosphoryl group in phospho-IIB^Chb may be completely ionized at pH 8.6. In this connection, it should be noted that the pK_a of the thiophosphoryl group in the IIB domain of S. carnosus could not be measured in the pH range 3.9-8.4 (18), and von Strandmann et al. concluded that the pK_s was likely to be <2.5 and that the phosphoryl group is doubly charged over a very broad pH range. There were no pH stability studies reported on this thiophosphoryl domain, and additionally, the IIB domain was subcloned with a His₆ tag to aid in the purification. Conceivably, the latter could influence the pK_a of the phosphoryl group in this protein.

The most unexpected result of the present studies, was to find that phospho-IIA^Chb and the mutant protein Cys10SerIIB^Chb form a stable complex, stable, that is, to gel filtration column chromatography, native gel electrophoresis, and analytical ultracentrifugation (some dissociation was noted). The complex was dissociated to its components by SDS-PAGE, and they were present in equimolar quantities in the complex as determined by densitometric scanning of the gels. The latter result does not distinguish between two possibilities, phospho-IIA^Chb/IIB^Chb and (phospho-IIA^Chb/IIB^Chb)₂, respectively, but the sedimentation studies show that it is a tetramer.

The complex was cross-linked with BS³, shown schematically in Fig. 7. The complex and the cross-linked complex are, we believe, analogues of a transition state complex that occur as an intermediate in the phosphotransfer reaction between IIA^Chb and IIB^Chb. Insofar as we are aware, no such transition state analogues involving protein-protein phosphotransfer reactions have been reported. Attempts are now in progress to crystallize the cross-linked analogue.

	FOOTNOTES

^* This work was supported by Grant 38759 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611.

^§ Present address: Zentrum Biochemie, OE 4310, Medizinische Hochschule Hannover, D-30623 Hannover, Germany.

^¶ To whom correspondence should be addressed: Dept. of Biology and the McCollum-Pratt Inst., Johns Hopkins University, Mudd Hall, Rm. 214, 3400 N. Charles St., Baltimore, MD 21218.

Published, JBC Papers in Press, July 25, 2000, DOI 10.1074/jbc.M001045200

¹ The subject matter of the accompanying manuscripts is as follows: (GlcNAc)₂ is a PTS sugar in E. coli (1); characterization of IIA^Chb from E. coli (2); analytical sedimentation studies on IIA^Chb, IIB^Chb, the phosphoproteins and a model transition state analogue (3); identification and molecular cloning of a chitoporin from Vibrio furnissii (4); and cloning and characterization of a (GlcNAc)₂ phosphorylase from V. furnissii (5).

	ABBREVIATIONS

The abbreviations used are: (GlcNAc)_n, -1,4-linked oligomers of GlcNAc where n = 2-6; PTS, phosphoenolpyruvate:glycose phosphotransferase system; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; PEP, phosphoenolpyruvate; BS³, bis(sulfosuccinimidyl)suberate; DTSSP, 3,3'-dithiobis(sulfosuccinimidyl proprionate).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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