Analytical Sedimentation of the IIAChb and IIBChb Proteins of the Escherichia coli N,N′-Diacetylchitobiose Phosphotransferase System

The phosphoenolpyruvate:glycose transferase system (PTS) is a prototypic signaling system responsible for the vectorial uptake and phosphorylation of carbohydrate substrates. The accompanying papers describe the proteins and product of theEscherichia coli N,N-diacetylchitobiose ((GlcNAc)2) PTS-mediated permease. Unlike most PTS transporters, the Chb system is composed of two soluble proteins, IIAChb and IIBChb, and one transmembrane receptor (IICChb). The oligomeric states of PTS permease proteins and phosphoproteins have been difficult to determine. Using analytical ultracentrifugation, both dephospho and phosphorylated IIAChb are shown to exist as stable dimers, whereas IIBChb, phospho-IIBChb and the mutant Cys10SerIIBChb are monomers. The mutant protein Cys10SerIIBChb is unable to accept phosphate from phospho-IIAChb but forms a stable higher order complex with phospho-IIAChb (but not with dephospho-IIAChb). The stoichiometry of proteins in the purified complex was determined to be 1:1, indicating that two molecules of Cys10SerIIBChb are associated with one phospho-IIAChb dimer in the complex. The complex appears to be a transition state analogue in the phosphotransfer reaction between the proteins. A model is presented that describes the concerted assembly and disassembly of IIAChb-IIBChb complexes contingent on phosphorylation-dependent conformational changes, especially of IIAChb.

quent sequence is as follows: phospho-Enzyme I donates its phosphate to HPr, phospho-HPr to sugar-specific IIA proteins, phospho-IIA to IIB, and phospho-IIB in conjunction with the membrane receptor, IIC, phosphorylates and transports the substrate. The structure and oligomeric state(s) of both Enzyme I and HPr have been extensively studied (for reviews see Refs. 6 -8). Several sugar-specific PTS transporters have also been well characterized, including the Escherichia coli glucose and mannitol transporters. To date, however, there have been few reports on the oligomeric states of the sugar-specific proteins, particularly in their phosphorylated states. Furthermore, although phosphate is transferred from one protein to another as summarized above, there have been no reports describing the isolation of a transition state intermediate or stable complex between two reacting PTS proteins.
In the accompanying papers (1-3), 2 we characterize the transport kinetics, product, and two of the sugar-specific transport proteins of the (GlcNAc) 2 (N,NЈ-diacetylchitobiose) or chb catabolic operon of E. coli. The phosphoryl transfer reaction sequence resulting in the uptake of (GlcNAc) 2 was studied, and the soluble proteins IIA Chb , IIB Chb , and an active site mutant (C10S), of IIB Chb were purified to apparent homogeneity. A phosphoryl group is transferred from phospho-HPr to IIA Chb and from phospho-IIA Chb to IIB Chb . Phospho-IIA Chb was found to be 5-10-fold more stable than homologous phospho-IIA proteins. This stability, as well as that of phospho-IIB Chb , enabled us to use analytical ultracentrifugation to determine the oligomeric states of IIA Chb and IIB Chb in both their unphosphorylated and phosphorylated forms and of an analogue of a potential transition state intermediate in the phosphotransfer reaction between the proteins.

EXPERIMENTAL PROCEDURES
Materials-Buffers and reagents were of the highest purity available. The solvent used in all analytical ultracentrifuge experiments was 25 mM sodium phosphate buffer, pH 8, unless otherwise specified. The values of solvent density and viscosity were calculated from composition as described in Laue et al. (9) using either the program SEDNTERP or Ultrascan.
Protein Samples-Purified proteins IIA Chb , IIB Chb , and Cys10SerIIA Chb , their respective phosphorylated forms, and phospho-IIA Chb -Cys10SerIIB Chb complex were prepared as described in the accompanying reports (2,3). Native gel and SDS-denaturing polyacryl-amide gel electrophoresis were performed using standard protocols. Protein bands were visualized using Coomassie Brilliant Blue and where indicated quantitated by densitometric scanning using the Eagle Eye II Still Video System and software (Stratagene). The partial specific volume (v) of each sample was estimated from the amino acid sequence using the method of Cohn and Edsall (10) as implemented in the program SEDNTERP (9).
Data Acquisition and Analysis-Ultracentrifugation experiments were performed in a Beckman XL-I analytical ultracentrifuge. All samples prepared for analytical ultracentrifugation experiments were in 25 mM sodium phosphate buffer, pH 8.0, unless otherwise noted. Samples were either dialyzed against the buffer prior to experiments or equilibrated with the buffer using gel filtration chromatography.
Velocity runs were conducted at either 4 or 20°C at 45,000 rpm in an An-60 rotor. Data scans were recorded continuously throughout the experiment using the absorption optical system at either 280 or 230 nm. Protein concentrations were adjusted to about 0.8 absorbance at the detection wavelength. Sample cells with 12-mm double sector charcoal filled epon centerpieces and quartz windows were used for all velocity runs. All data were analyzed using the method of van Holde and Weischet (11,12) as implemented in the program Ultrascan. Following preliminary analysis, data were further fitted using the finite element method of Demeler and Saber (13) also as implemented in Ultrascan.
Equilibrium runs were conducted at 20°C using either standard cells or short column eight channel cells equipped with sapphire windows. Several loading concentrations and several speeds were used for each sample. Data were collected using the absorption optical system at either 280 or 230 nm for runs in standard cells and with the interference optical system for short column runs. All sedimentation equilibrium data were analyzed using the program NONLIN (14).

RESULTS
Velocity Sedimentation Analyses of IIA Chb , Phospho-IIA Chb , IIB Chb , Phospho-IIB Chb , and Cys10SerIIB Chb -Samples for velocity sedimentation were prepared as described under "Experimental Procedures." Phosphorylated forms of the indicated proteins were used immediately after preparation. Phosphorylation was monitored both before and after centrifuge runs using the gel shift mobility assay (2). Total phosphorylation was at least 95% in each sample, and unless otherwise specified, less than 10% dephosphorylation occurred during the time of the run.
Sedimentation velocity data on IIA Chb , phospho-IIA Chb , IIB Chb , phospho-IIB Chb , and the active site mutant Cys10SerIIB Chb were collected at 20°C, and analyzed by the method of van Holde and Weischet (11,12). For each protein, an extrapolation plot of S apparent versus 1/ ͱ t across the boundary converged to a single y intercept indicating homogeneity of the samples. Integral distribution plots, G(s), of the diffusion-corrected sedimentation coefficient versus boundary fraction are shown in Fig. 1 for all five samples. IIB Chb and Cys10SerIIB Chb show essentially identical sedimentation behavior, indicating that there is no significant hydrodynamic change introduced by the mutation. Phosphorylation of IIB Chb leads to a 20% increase in s 20,w . IIA Chb has a much higher s 20,w than IIB Chb , and in contrast to IIB Chb , phosphorylation of IIA Chb results in about a 15% decrease in the sedimentation coefficient.
To derive the hydrodynamic parameters for each protein and better understand the implications of these changes in s 20,w , all data were fitted directly to the Lamm equation using the finite element fitting method as implemented in the program Ultrascan. A summary of the fitted sedimentation and diffusion coefficients, calculated molecular weights, hydrodynamic radii, and frictional coefficients are given in Table I. Given that the known molecular weight of the IIA Chb dimer is about 25,496, the results indicate that IIA Chb is a stable dimer. Because no monomeric species were detected, the upper limit of the K d is 10 Ϫ7 M (15). Additionally, no monomeric species were detected after phosphorylation of IIA Chb , even though the s 20,w value for phospho-IIA Chb was 10 -15% lower than for the unphosphorylated protein. This observation was consistent and reproducible and suggests that phosphorylation of IIA Chb induces a more elongated conformation. Furthermore, samples that were cycled between the phosphorylated and dephosphorylated states showed a reproducible shift between the corresponding low and high s values when analyzed by sedimentation velocity centrifugation (data not shown).
Possible nonideality effects because of the low ionic strength of the solvent (25 mM NaPO 4 , pH 8.0) were investigated by repeating the experiments in the same buffer containing 0.1 M NaCl. Similar results as those summarized in Table I were obtained. Although there was a slight change in the calculated s value for each species, the s 20,w value for IIA Chb dropped 10 -15% upon phosphorylation, and finite element fitting indicated that both species are stable dimers (data not shown). The estimated s 20,w values were also found to be dependent on the temperature of the experiment. For runs carried out at 4°C, s 20,w for IIA Chb and phospho-IIA Chb were found to be nearly identical (Table I), whereas they were different at 20°C. Here again, fitting of the data showed both species to be dimeric.
In contrast to the results obtained with IIA Chb and its phospho derivative, a comparison of the known molecular masses with those obtained by finite element fitting of the velocity sedimentation data indicated that IIB Chb , phospho-IIB Chb , and the mutant Cys10SerIIB Chb sediment as monomers under these conditions. Equilibrium Sedimentation Analysis of IIA Chb , IIB Chb , and Cys10SerIIB Chb -Equilibrium sedimentation data were collected for IIA Chb , IIB Chb , and Cys10SerIIB Chb in standard double sector cells with a 3-mm column (Fig. 2). Samples of these proteins were brought to equilibrium at three initial loading concentrations and three speeds (for IIA Chb ) or five speeds (for IIB Chb and Cys10SerIIB Chb ). The data were analyzed by global FIG. 1. Integral distribution (G(s)) plots of data from sedimentation velocity experiments with IIA Chb , phospho-IIA Chb , IIB Chb , phospho-IIB Chb , and Cys10SerIIB Chb . Data were obtained using the scanning absorption optics of either a Beckman XL-A or XL-I analytical ultracentrifuge. Experiments were performed at 20°C for all samples except phospho-IIB Chb , which was done at 4°C to minimize dephosphorylation during the course of the run. All data were collected at 230 nm where the initial sample absorbances were 0.7-0.9. All runs were carried out in a solvent comprising 25 mM sodium phosphate buffer, pH 8.0, at 20°C. Derived sedimentation coefficients have been corrected to 20°C in H 2 O. Integral distribution plots of data gathered on purified proteins are indicated as follows: IIA Chb (E), phospho-IIA Chb (q), IIB Chb (Ⅺ), phospho-IIB Chb (f), and Cys10SerIIB Chb (‚). Open symbols represent unphosphorylated samples, and closed symbols represent phosphorylated samples.
fitting to a single ideal species model, and the results are summarized in Table II. In close agreement with the velocity sedimentation results, sedimentation equilibrium data indicate that IIA Chb exists as a dimer, whereas both IIB Chb and Cys10SerIIB Chb are monomers at the concentrations used in these experiments.
Short Column Equilibrium Sedimentation Analysis of IIA Chb and Phospho-IIA Chb -Using a standard column height, equilibrium sedimentation data could not be gathered for the phosphorylated form of IIA Chb because of the lability of the phosphate group during the several days required for a typical equilibrium run. To circumvent this limitation, we conducted short column equilibrium studies with both IIA Chb and phospho-IIA Chb . This permitted equilibrium data to be obtained at three speeds, using four loading concentrations each, in less than 5 h. The data were globally fitted to a single ideal species model using the program NONLIN, and results are summarized in Table II. Results with IIA Chb are fully consistent with the standard sedimentation equilibrium results described above. Similarly, short column data with phospho-IIA Chb are well described by a single ideal species model with a molecular weight indicative of a dimer.
Velocity Sedimentation Analysis of Mixtures of IIA Chb and IIB Chb -Attempts were made to determine whether complexes could be detected in mixtures of IIA Chb and IIB Chb and/or their phospho derivatives. Equimolar quantities of the protein pairs were mixed and maintained at room temperature until loaded into the centrifuge (30 -60 min). Under these conditions phospho-transfer can occur between the proteins, until presumably an equilibrium is reached.
The following mixtures were tested: IIA Chb and IIB Chb , phospho-IIA Chb and IIB Chb , IIA Chb and phospho-IIB Chb , and IIA Chb and Cys10SerIIB Chb . Extrapolation plots indicated that each data set was composed of a mixture of the two components with s values similar to those of the individual protein species. Diffusion corrected integral distribution plots for various mixtures are presented in Fig. 3. Finite element fitting of the data to a noninteracting two species model in each case gave sedimentation coefficients consistent with those of the individual proteins. 3 Thus, we concluded that none of the protein pairs tested formed significant concentrations of stable higher order complexes, i.e. complexes detectable by analytical sedimentation.
In sharp contrast to the results obtained above, when a sample containing the mutant Cys10SerIIB Chb , which cannot accept the phosphoryl group, was incubated with phospho-IIA Chb , a higher order complex of approximately 4.2 s was observed (Fig. 3). As indicated above, this species was not detected in the mixture of Cys10SerIIB Chb and (unphosphorylated) IIA Chb . These results indicate that a complex between phospho-IIA Chb and IIB Chb is stabilized under conditions where phosphotransfer cannot occur. Short Column Equilibrium Sedimentation Analysis of Phospho-IIA Chb and Cys10SerIIB Chb -To verify the sedimentation velocity results above and to determine the molecular weight of the complex, it was desirable to obtain equilibrium data on the mixture of phospho-IIA Chb and Cys10SerIIB Chb . The instability of the phosphate group in the phospho-IIA Chb , even at lowered temperatures, precluded standard equilibrium experiments because of the time required to make the measurements. It is, however, possible to gather data on the complex using a short solution column. Short column data on a stoichiometric mixture of phospho-IIA Chb and Cys10SerIIB Chb were obtained at four initial loading concentrations and two speeds in about 5 h (Fig. 4). These data were fitted to a single ideal species model and yielded an estimate on the molecular weight of the complex of 55,600. The best fit lines and combined residuals for the fit versus both the dependent and independent variable are also presented. Attempts to fit these data to more complex models yielded no significant improvement in the fit.
Stoichiometry of Phospho-IIA Chb and Cys10SerIIB Chb in the Complex-Although the velocity centrifugation data clearly showed the formation of a complex between phospho-IIA Chb and Cys10SerIIB Chb , the estimated molecular weights did not allow discrimination between a ternary and a quaternary complex. That is, it could be either phospho-IIA Chb dimer and 1 mol of Cys10SerIIB Chb (molecular mass, 36 kDa) or phospho-IIA Chb dimer and 2 mol of Cys10SerIIB Chb (molecular mass, 48 kDa). The short column equilibrium data gave a molecular weight 55,631 (Ϯ 1870) consistent with a complex of one phospho-IIA Chb dimer and either two IIB Chb monomers (48,000) or possibly three IIB Chb monomers (60,000).
To independently determine the stoichiometry of the proteins in the complex, the purified complex was subjected to denaturing SDS-polyacrylamide gel electrophoresis, and protein bands were quantitated and compared with known amounts of each individual protein as standards (3). The results showed that the complex was composed of equimolar quantitities of phospho-IIA Chb monomer and the mutant protein Cys10SerIIB Chb . We therefore concluded that the complex is a tetramer, composed of 1 mol of a dimer of phospho-IIA Chb and 2 mol of Cys10SerIIB Chb . DISCUSSION There are only a few studies on the hydrodynamic properties of the sugar-specific PTS proteins, and insofar as we know, none on the corresponding phosphoproteins (6, 7). In the present experiments, analytical sedimentation, both velocity and equilibrium, were used to characterize the following proteins of the N,NЈ-diacetylchitobiose transport system: IIA Chb , phospho-IIA Chb , IIB Chb , phospho-IIB Chb , a mutant Cys10SerIIB Chb ), and an apparent complex between phospho-IIA Chb and the mutant protein. The latter is a likely candidate as a transition state analogue in the phosphotransfer reaction between IIA Chb and IIB Chb . IIA Chb has considerable amino acid sequence similarity to IIA Lac , part of the lactose PTS permease in Gram-positive organisms; IIA Chb is 33% identical to IIA Lac of Staphylococcus aureus and 35% identical to the same protein from Lactococcus lactis (7,16). The crystal structure of the latter protein reveals that it is 83% ␣-helix (16), whereas in the accompanying paper (2) we report that IIA Chb is 75-85% helix. Nevertheless, IIA Chb is very different from IIA Lac . Sedimentation equilibrium experiments (17) clearly established that IIA Lac forms a stable trimer, whereas the results reported here show that IIA Chb forms a very stable dimer. Because no monomer was detected, the dissociation constant for the dimer would have to be less than 10 Ϫ7 M (15).
Further, there are other major differences between phospho-IIA Lac and phospho-IIA Chb . IIA Lac is thought to dissociate when it is phosphorylated (18). No monomeric phospho-IIA Chb was detected in the sedimentation experiments reported here.
Although they have the same molecular weight, the sedimentation coefficient of phospho-IIA Chb was 10 -15% less than that of IIA Chb , suggesting that phosphorylation yielded a significantly less compact protein. These results agree with those obtained from the CD spectra, where it appeared that at 37°C, phospho-IIA Chb loses ϳ35% of the helicity of IIA Chb (2) and was much more sensitive to thermal denaturation. The effects of protein concentration both on the stability to hydrolysis of the phosphoprotein and on its thermal denaturation suggested that the phosphodimer dissociates to phosphomonomer, but this effect was only apparent at 37°C and above. The sedimentation experiments were conducted at temperatures ϳ20°C because significant hydrolysis of the phosphoprotein does occur over prolonged periods at the higher temperatures. The sedimentation velocity data indicate a change in s 20,w for IIA Chb but not for phospho-IIA Chb as a function of temperature (Table I). Because no change in extent of dimerization is observed, this result suggests an important change in shape and/or hydration with temperature. We are currently investigating this observation in greater detail.
The solution and crystal structures of the mutant protein Cys10SerIIB Chb have been described (19,20), but there are no reports on the properties of IIB Chb nor phospho-IIB Chb . The sedimentation results presented here show that: (a) IIB Chb , phospho-IIB Chb , and Cys10SerIIB Chb each behaves as a single monomeric species. (b) IIB Chb and the mutant Cys10SerIIB Chb exhibited identical sedimentation behavior, therefore suggesting that this mutation causes no significant change in structure  C, 40,200, 45,000, 49,400, 57,000, and 60,000 rpm). All data were globally fit to a single ideal species model using the program NONLIN. The fitted curves are superimposed on the data in the main chart, and the residuals of the fit are plotted above the main chart (versus the independent variable). A summary of the fitted molecular weights is presented in Table II. This run was performed using cells with 12-mm double-sector charcoal filled epon centerpieces and quartz windows. Equilibrium was established when data obtained from scans taken 2 h apart were indistinguishable. The solvent comprised 25 mM sodium phosphate buffer, pH 8, and the run temperature was 20°C.
(as reflected in the hydrodynamic properties). In contrast to IIA Chb , where phosphorylation resulted in a lower sedimentation coefficient, phospho-IIB Chb exhibited a significantly higher coefficient than IIB Chb , implying that the phosphoprotein may be more compact than unphosphorylated IIB Chb . (Similar effects can result from changes in hydration and/or shape.) As discussed elsewhere in these papers (3), phospho-IIB Chb is a thiophosphoryl protein, analogous to protein-tyrosine phosphate phosphatases. In the latter, phosphorylation at the active site Cys causes a conformationally flexible loop in the peptide chain to close over the phosphoryl group, which conceivably would yield a more compact protein with a higher sedimentation coefficient. Finally, it should also be noted that IIB Chb is a basic protein (pI about 8.0) and that the phosphoryl group may interact with basic groups in the protein, thereby resulting in a more compact structure than IIB Chb .
When phospho-IIA Chb was mixed with an equimolar quantity of Cys10SerIIB Chb , a new molecular species was observed that was stable to both native gel electrophoresis and to gel filtration chromatography (3). No other combination of proteins gave this result. Fig. 3 presents sedimentation velocity data using the following mixtures of proteins: IIA Chb and IIB Chb ; phospho-IIA Chb and IIB Chb ; IIA Chb and phospho-IIB Chb ; and IIA Chb and Cys10SerIIB Chb . These four combinations of proteins exhibited sedimentation coefficients predicted from the data obtained with the individual species, i.e. no higher order species were observed, indicating that none of the protein pairs  3. Integral distribution plots of stoichiometric mixtures of IIA Chb and IIB Chb , phospho-IIA Chb and IIB Chb , IIA Chb and phospho-IIB Chb , IIA Chb and Cys10SerIIB Chb , and phospho-IIA Chb and Cys10SerIIB Chb . Sedimentation velocity data were collected with different equimolar protein mixtures. Experimental conditions are the same as described in the legend to Fig. 1. Integral distribution plots of IIA Chb and IIB Chb (Ⅺ), phospho-IIA Chb andIIB Chb (q), IIA Chb and phospho-IIB Chb (f), IIA Chb and Cys10SerIIB Chb (‚), and phospho-IIA Chb and Cys10SerIIB Chb (OE) are shown. Open symbols represent unphosphorylated samples, and closed symbols represent samples with one component phosphorylated.
FIG. 4. Short column equilibrium sedimentation data of equimolar mixture of phospho-IIA Chb and Cys10SerIIB Chb . Sedimentation equilibrium data were collected on an equimolar mixture of phospho-IIA Chb and Cys10SerIIB Chb . The chart shows eight concentration distributions collected from samples at four loading concentrations (2.5, 1.25, 0.625, and 0.31 mg/ml) and two speeds (20,000 and 30,000 rpm). All data were globally fit to a single species model using the program NONLIN. The fitted curves are superimposed on the data in the main chart, and the residuals of the fit are plotted above the main chart (versus the independent variable) and to the right of the main chart (versus the dependent variable). This run was performed using a short column 8-channel centerpiece to minimize the time required to reach equilibrium. The cell was equipped with sapphire windows, and all data were collected using the interference optics of the XL-I. Scans were taken every 15 min until no change in the fringe pattern was detectable, about 90 min at each speed. The solvent comprised 25 mM sodium phosphate buffer, pH 8, and the run temperature was 20°C.
formed a stable complex. (Because phosphoryl transfer does occur between two of the pairs of proteins, a transient complex, at least, must be formed, but its concentration was either too low to be detected or the transfer was complete prior to sedimentation.) By sharp contrast to these results, an equimolar mixture of phospho-IIA Chb and Cys10SerIIB Chb yielded a new species of much higher sedimentation coefficient than each of the individual proteins. A short column sedimentation equilibrium experiment revealed that the complex had about the expected molecular weight for a tetramer, which agreed with the analyses (3). The composition of the complex is therefore: 1 mol of phospho-IIA Chb dimer and 2 mol of Cys10SerIIB Chb . The dissociation constant of the complex must be less than 10 Ϫ7 M (15). Thus, we have been able to characterize a complex formed between phospho-IIA Chb and the mutant, Cys10SerIIB Chb . As far as we have been able to determine, there are no previous reports on transition state analogue intermediates in a phosphotransfer reaction between two proteins.
A model depicting our conclusions of these and related CD spectral studies is schematically depicted in Fig. 5. Phosphorylation of the IIA Chb dimer results in a conformational change that permits binding of two mols of IIB Chb to form one or more transient transition state complexes. Phosphate transfer to IIB Chb and dissociation to the products completes the reaction, with concomitant change of IIA Chb back to its original conformation.

FIG. 5. Model of interaction between phospho-IIA Chb and IIB Chb .
The IIA Chb dimer is phosphorylated by phospho-HPr. The model depicts a change in conformation of the dimer when it is phosphorylated, consistent with the sedimentation and CD spectral data. The phosphoprotein binds two molecules of IIB Chb to form a transient complex, which dissociates when the phosphoryl group is transferred to IIB Chb , and the IIA Chb dimer returns to its original conformation.