Thermodynamic Evidence for Conformational Coupling between the B and C Domains of the Mannitol Transporter of Escherichia coli, Enzyme IImtl*

Wim Meijberg, Gea K. Schuurman-Wolters, and George T. RobillardDagger

From the Groningen Biomolecular Sciences and Biotechnology Institute and the Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The transport across the cytoplasmic membrane and concomitant phosphorylation of mannitol in Escherichia coli is catalyzed by the mannitol-specific transport protein from the phosphoenolpyruvate-dependent phosphotransferase system, enzyme IImtl. Interactions between the cytoplasmic B and the membrane embedded C domain play an important role in the catalytic cycle of this enzyme, but the nature of this interaction is largely unknown. We have studied the thermodynamics of binding of (i) mannitol to enzyme IImtl, (ii) the substrate analog perseitol to enzyme IImtl, (iii) perseitol to phosphorylated enzyme IImtl, and (iv) mannitol to enzyme IImtl treated with trypsin to eliminate the cytoplasmic domains. Analysis of the heat capacity increment of these reactions showed that approximately 50-60 residues are involved in the binding of mannitol and perseitol, but far less in the phosphorylated state or after removal of the B domain. A model is proposed in which binding of mannitol leads to the formation of a contact interface between the two domains, either by folding of unstructured parts or by docking of preexisting surfaces, thus positioning the incoming mannitol close to the phosphorylation site on the B domain to facilitate the transfer of the phosphoryl group.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The transport of carbohydrates from the environment into the bacterial cell is, in many cases, accomplished by a complex of proteins that together make up the phosphoenolpyruvate-dependent phosphotransferase system (PTS)1 (1, 2). This system consists of a number of transport proteins, each one specific for one carbohydrate, and a chain of cytoplasmic proteins that ultimately transfer a phosphoryl group derived from PEP to the incoming carbohydrate. The first protein in this chain is enzyme I, which accepts the phosphoryl group from PEP and transfers it to HPr, which in its phosphorylated form is the substrate for the transport proteins. All transport proteins of the PTS have a similar architecture, consisting of a part that binds and transports the carbohydrate and is embedded in the cytoplasmic membrane and two cytoplasmic parts, responsible for the phosphorylation of the substrate. The parts may or may not be covalently bound, and all possible combinations do, in fact, exist. In the case of the mannitol-specific transporter from Escherichia coli, termed enzyme IImannitol, all three parts are covalently linked and are, thus, domains of the same protein. The phosphoryl group is accepted from P-HPr by His-554 in the C-terminal A domain and transferred to Cys-384 on the B domain. Mannitol is bound at the periplasmic side and translocated across the membrane by the N-terminal C domain, after which the carbohydrate is phosphorylated by the B domain. The protein is believed to function as a dimer, both in the membrane and solubilized in detergent (3-9).

It is obvious from the above that domain interactions play an important role in the catalytic cycle of this enzyme. Lolkema et al. (10), for instance, have shown that phosphorylation of the B domain causes a 1000-fold increase in the rate of isomerization of the mannitol-occupied C domain, the principle event in the transport process. Further evidence that the B and C domains influence each other's conformation was obtained from an analysis of the binding kinetics of mannitol to the wild-type enzyme, the subcloned C domain, and a number of mutants substituted at the position of the second phosphorylation site (10, 11). However, in the absence of knowledge of the three-dimensional structure in the intact complex, the nature of the interaction is still largely unknown. Thermodynamic data can give additional information, both on the nature of the interaction and also on the extent of the structural changes that are involved. We have chosen isothermal titration calorimetry (ITC) in this study to develop the required thermodynamic data.

In this study, we determine the energetics of mannitol binding to EIImtl by ITC in inside-out membrane vesicles, i.e. under conditions that closely resemble the native state of the protein. To study the effect of phosphorylation, the binding of a substrate analog, perseitol, which cannot be transported or phosphorylated (12), to EIImtl in the phosphorylated and unphosphorylated state is determined and compared with binding of mannitol. Finally, the effect of removal of the A and B domains on the functioning of the C domain is investigated. In all of these cases, the experiments have been performed at various temperatures, enabling us to calculate Delta Cpoobs. This parameter has been shown to correlate with the change in solvent-exposed groups before and after the binding reaction (see, e.g., Refs. 13-15) and can, therefore, be used to monitor structural rearrangements taking place in the enzyme upon binding of the substrate.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- D-[1-14C]Mannitol (2.04 GBq/mmol) was obtained from Amersham Pharmacia Biotech; D-[1-3H]mannitol (976.8 GBq/mmol) was from NEN Life Science Products. Perseitol was obtained from Pfanstiehl Laboratories Inc., Waukagan, IL. TPCK-treated trypsin was from Worthington or Sigma, and bovine pancreatic trypsin inhibitor was from Sigma. Enzyme I and HPr were purified as described previously (16, 17). Decylpoly(ethylene glycol) 300 was synthesized by B. Kwant at the Department of Chemistry, University of Groningen. All other chemicals were of the highest purity available.

Growth of Bacteria and Preparation of Membrane Vesicles-- Membrane vesicles containing large amounts of EIImtl were obtained from E. coli strain LGS322 (F- thi-1, hisG1, argG6, metB1, tonA2, supE44, rpsL104, lacY1, galT6, gatR49, gatA50, Delta (mtlA'p), mtlDc, D(gutR'MDBA-recA)), which contains a chromosomal deletion for the wild type mtlA gene, transformed with the inducible plasmid pMamtlA (18), encoding for EIImtl. A single colony was used to inoculate 10 ml of LB medium (10 g/liter Bactotryptone, 5 g/liter Bactoyeast extract, 10 g/liter NaCl) containing 100 µg/ml ampicillin. This culture was grown at 30 °C to an A600 of approximately 0.6 and then used to inoculate a 1.5-liter culture in the same medium, which was grown overnight to an A600 of approximately 3. This was used to inoculate a 60-liter fermenter containing the same medium. The cells were grown to an A600 of 0.7, induced for 2 h at a temperature of 42 °C and harvested. The final A600 was 0.85, resulting in a total of 86 g of cells (wet weight). The cells were washed three times with ~2 liters of buffer (50 mM NaPi, pH 7.5, 1 mM NaN3) and stored on ice overnight. From this preparation, inside-out membrane vesicles were obtained as described by Lolkema and Robillard (8). The final yield was 100 ml of vesicle solution containing 30 µM EIImtl. The preparation was stored in 2-ml aliquots at -80 °C until use. Approximately 30% of the total protein was EIImtl, analyzed by SDS-polyacrylamide gel electrophoresis. The orientation of the protein with respect to the membrane was determined from the mannitol phosphorylation activity before and after treatment with trypsin (5) and turned out to be approximately 90% inside-out.

Membrane vesicles not containing EIImtl were prepared from E. coli LGS322 without the plasmid. In this case, 9 liters of LB medium (without ampicillin) was inoculated 1:50 and grown to an A600 of 0.9 and then induced in the manner described above. Membrane vesicles were prepared as described for the cells containing the plasmid.

PEP-dependent Mannitol Phosphorylation-- The PEP-dependent phosphorylation of mannitol by EIImtl was determined as described (19).

Concentration Determination-- The concentration of EIImtl in the vesicles was determined by the mannitol burst method, which quantitates the amount of phosphorylation sites present in the preparation (20). 20 nM enzyme I and 1 µM HPr were added to three aliquots of 160 µl of a standard buffer solution (25 mM Tris-HCl, pH 8.3, 5 mM DTT, 5 mM MgCl2, 5 mM PEP, 7 mM decylmaltoside). Under these conditions, the kinetics of conversion of mannitol to mannitol-1-P are slow and limited by the transfer of the phosphoryl group from P-HPr to the A domain of EIImtl. A 10 mM [3H]mannitol solution (20 µl) was added to one aliquot. In this case, the mannitol phosphorylation reaction was started by adding 40 µl of the vesicle solution and monitored by applying 20-µl aliquots to a small Dowex column at 20-s intervals as described (19). A vesicle solution (40 µl) was added to each of the other two aliquots and incubated at 30 °C, one for 10 and the other for 30 min to allow for complete phosphorylation of EIImtl. The assay was then started by adding 20 µl of the mannitol solution to each mixture and monitored in the same way. No differences were observed between the samples incubated for 10 and 30 min in the absence of mannitol, indicating that phosphorylation of EIImtl is completed within 10 min. The change in the concentration of mannitol-1-P in time was extrapolated back to t = 0 to determine the difference in the mannitol-1-P concentration between samples incubated with and without PEP. This difference equals the concentration phosphorylation sites on EIImtl in the preparation, assuming that the transfer of all the EIImtl phosphoryl to mannitol is rapid and complete. Since EIImtl contains two phosphorylation sites per monomer, this number is divided by two to obtain the concentration EIImtl.

Isothermal Titration Calorimetry-- All titrations were performed using a MCS isothermal titration calorimeter from Microcal. A similar instrument has been described elsewhere (21). During experiments, the vesicle solution was thermostatted at the desired temperature and stirred at 700 rpm. The injection sequence consisted of 30 injections of 3 µl each from a 100-µl syringe, unless indicated otherwise. Data were analyzed using Origin software from Microcal.

Samples containing vesicles were dialyzed overnight against 1 liter of buffer (50 mM KPi or Tris/HCl, pH 7.5, 2 mM EDTA, 1 mM DTT) and, if necessary, diluted in the dialysis buffer before the experiment. Mannitol and perseitol solutions were prepared from stock solutions in H2O (30.69 and 13.64 mM, respectively) by dilution in dialysis buffer to minimize artifacts arising from mixing the two different buffer solutions during the ITC experiment. Control experiments in which the mannitol or perseitol solution was injected into the buffer were routinely run and always showed negligible peaks. All solutions were degassed by gently stirring under vacuum directly before measurements.

To study the interaction of perseitol with phosphorylated EIImtl, the procedure was slightly modified. After dialysis overnight, 200 ml of the dialysis buffer was taken and brought to 7 mM in MgCl2 and 2 mM in PEP. At the same time, small volumes of a 80 µM HPr and 100 mM PEP solution were added to 2 or 4 ml of the vesicles solution to give final concentrations of 5.5 µM and 2 mM, respectively. The vesicle solution was then reequilibrated with the modified buffer by dialysis for 4 h. Before calorimetric experiments, a small volume of a 3.3 µM enzyme I solution was added to the vesicles solution to yield a final enzyme I concentration of 120 nM. The solution was incubated at room temperature for 10 min before loading the sample into the calorimeter. Because of the low enthalpy of binding in this reaction, the injection sequence was changed to 16 injections of 6 µl each.

Vesicles containing only the C domain in the inside-out orientation were prepared from EIImtl-containing vesicles by proteolysis, using 10 µg/ml TPCK-treated trypsin for 45 min at room temperature. To stop the reaction, soybean trypsin inhibitor was added to a final concentration of 100 µg/ml, after which the normal dialysis procedure was followed. The success of the treatment was evaluated by monitoring mannitol phosphorylation activity and by SDS-polyacrylamide gel electrophoresis. Intact EIImtl runs at an apparent molecular mass of 56 kDa and the C domain at 30 kDa.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Binding of Mannitol to EIImtl-- Fig. 1A shows a typical example of the raw data obtained from a titration of EIImtl-containing inside-out vesicles with mannitol. Upon addition of small aliquots of the mannitol solution, exothermic signals are observed that, after saturation of the binding, become small and constant. For comparison, a titration of a solution of a similar concentration of vesicles prepared from E. coli LGS322 cells without the plasmid encoding for EIImtl is also shown. The resulting signals are small and constant, indicating the lack of specific binding of mannitol in this case. We can, therefore, conclude that the observed effect is due to the specific binding of mannitol to EIImtl.


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Fig. 1.   Raw titration calorimetry data. A, 33 injections of 3 µl each of a 300 µM mannitol solution into a solution of 15 µM EIImtl in inside-out membrane vesicles in 50 mM potassium phosphate, pH 7.5, 2 mM EDTA, 1 mM DTT at 20 °C (upper trace). For comparison, 13 injections of the same mannitol solution into a solution of membrane vesicles without EIImtl are also shown (lower trace). The peaks result from mixing of the two solutions. B, integrated heats of injection for the experiment shown in the top panel. The solid line represents the best fit of the one-set-of-sites model to the data. The parameters derived from the fit are: n = 0.67 ± 0.01, Kd = 115 ± 17 nM, and Delta Hoobs = -63.4 ± 0.9 kJ mol-1.

The integrated heats for each injection were calculated from the areas under the peaks and, after correction for the heat of dilution of the mannitol solution, fitted to a mathematical binding model assuming one set of sites (Fig. 1B). The model uses three parameters: the stoichiometry of the reaction n, the association constant Ka, and the enthalpy change associated with binding Delta Hoobs (21). The heat resulting from the first injection often was somewhat smaller than heats generated from subsequent injections, probably due to imperfect filling or dilution of the mannitol solution at the tip of the syringe during equilibration of the calorimeter, and was, therefore, not taken into account during data analysis. The model fits the data well and yields an estimate of -63.4 ± 0.9 kJ mol-1 for Delta Hoobs, 115 ± 17 nM for the dissociation constant Kd, and 0.67 ± 0.01 for the stoichiometry of the reaction at 20 °C under the conditions given in the figure legend.

The heat capacity increment of the binding reaction, Delta Cpoobs, can be obtained from the temperature dependence of the enthalpy of binding. Fig. 2 shows the results of titrations of EIImtl with mannitol at various temperatures. The calculated thermodynamic parameters obtained from these data are listed in Table I. The variation of Delta Hoobs as a function of temperature is linear over the temperature interval that we have investigated (Fig. 2, inset), resulting in a value of -4.0 ± 0.3 kJ K-1 mol-1 for Delta Cpoobs in phosphate buffer.


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Fig. 2.   Integrated heats of injection for the titration of EIImtl with mannitol in 50 mM potassium phosphate, pH 7.5, 2 mM EDTA, 1 mM DTT at 12.6 °C (bullet ), 14.9 °C (open circle ), 20.0 °C (×), and 24.9 °C (black-square). The solid lines represent the best fit of the one-set-of-sites model to the data. Inset, the variation of Delta Hoobs with temperature. The solid line is obtained from linear least squares analysis and has a slope of -4.0 ± 0.3 kJ K-1 mol-1.

                              
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Table I
Mannitol binding by EIImtl
Parameters are derived from a nonlinear least squares fit to the data, using the one-set-of-sites model. Errors are the standard deviations obtained from the fits.

In general, there are two terms that contribute to Delta Hoobs: a term arising directly from the binding reaction and a term rising from a change in protonation state of the protein in the complexed and uncomplexed state.
&Dgr;H<SUP>o</SUP><SUB><UP>obs</UP></SUB>=&Dgr;H<SUP>o</SUP><SUB>0</SUB>+N<SUB><UP>H+</UP></SUB> &Dgr;H<SUP>b</SUP><SUB><UP>ion</UP></SUB> (Eq. 1)
NH+ is the number of protons released by the buffer and Delta Hbion is the ionization enthalpy of the buffer (5.3 kJ mol-1 for phosphate buffer; Ref. 22). To determine whether protons are released or taken up by the buffer as a result of the binding process, the experiment was repeated in 50 mM Hepes and Tris buffers at pH 7.5 (Delta Hbion 21 and 47.5 kJ mol-1, respectively). The binding of mannitol becomes less exothermic when Delta Hbion increases (Table I), indicating that protons are released to the buffer, with a calculated value for NH+/mol of mannitol of 0.45 ± 0.1 at 20 °C. The Delta Cpoobs for the interaction of EIImtl with mannitol in Tris buffer is -3.4 ± 0.4 kJ K-1 mol-1.

The Binding of Perseitol to EIImtl-- Perseitol is a structural analogue of mannitol (Fig. 3) that binds to EIImtl, but is not phosphorylated or transported across the membrane (12, 23). It inhibits the transport activity of the enzyme competitively, indicating that the binding sites for mannitol and perseitol are the same. The results of ITC experiments in which EIImtl is titrated with perseitol at temperatures between 10.5 and 30 °C are presented in Fig. 4 and Table II. The binding of perseitol can also be described by a one-set-of-sites model, using parameters that differ very little from the parameters that were obtained for the binding of mannitol to EIImtl. The apparent Kd for perseitol binding is larger by a factor of 1.5-2 compared with mannitol, whereas the Delta H of binding is about 5 kJ mol-1 less exothermic over the entire temperature interval. Delta Cpoobs for binding of perseitol is -3.9 ± 0.2 kJ K-1 mol-1, very close to the value for mannitol binding under the same conditions. It, therefore, seems that perseitol is a good model compound to study the changes in binding affinity upon phosphorylation of EIImtl, since the protein can not be dephosphorylated by perseitol.


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Fig. 3.   Mannitol (left) and perseitol (right). Both molecules are shown in Fisher projection.


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Fig. 4.   Integrated heats of injection for the titration of EIImtl with perseitol in 50 mM potassium phosphate, pH 7.5, 2 mM EDTA, 1 mM DTT at 10.5 °C (bullet ), 15.0 °C (open circle ), 20.0 °C (×), 25.3 °C (black-square), and 30.0 °C (square ). The solid lines represent the best fit of the one-set-of-sites model to the data. Inset, the variation of Delta Hoobs with temperature. The solid line is obtained from linear least squares analysis and has a slope of -3.9 ± 0.2 kJ K-1 mol-1.

                              
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Table II
Perseitol binding by EIImtl
Parameters are derived from a nonlinear least squares fit to the data, using the one-set-of-sites model. Errors are the standard deviations obtained from the fits. All data were recorded in 50 mM KPi, pH 7.5, 2 mM EDTA, 1 mM DTT.

The Binding of Perseitol to Phosphorylated EIImtl-- The binding of perseitol to the phosphorylated state of EIImtl proved to be difficult to follow by ITC because we observed only a small enthalpy change associated with the reaction, resulting in a low signal to noise ratio. In addition, measurements at temperatures above 25 °C were hampered by instability of the base line, possibly due to aggregation during the experiments. Nonetheless, we were able to determine the binding parameters for the interaction at two different temperatures, allowing us to roughly estimate Delta Cpoobs (Fig. 5 and Table III). Phosphorylated EIImtl binds perseitol with an affinity comparable to the unphosphorylated enzyme, but the contributions of Delta Hoobs and Delta Soobs to the free energy of binding differ considerably in both cases. In the case of the non-phosphorylated enzyme, there is a much larger contribution of the enthalpy of binding to the stabilization of the complex than when the enzyme is in its phosphorylated state. This is compensated for, however, by a larger contribution of the entropy term, an effect that has been termed enthalpy-entropy compensation and has been observed for a large number of non-covalent interactions (see "Discussion"). Delta Cpoobs of the binding reaction of perseitol with phosphorylated EIImtl is less than 1 kJ K-1 mol-1, both in phosphate and Tris buffers, i.e. less than 25% of the value for the non-phosphorylated enzyme.


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Fig. 5.   Integrated heats of injection for the titration of phosphorylated EIImtl with perseitol in 50 mM potassium phosphate, pH 7.5, 2 mM EDTA, 1 mM DTT at 20.5 °C (bullet ) and 25.3 °C (open circle ). The solid lines represent the best fit of the one-set-of-sites model to the data.

                              
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Table III
Perseitol binding by phosphorylated EIImtl
Parameters are derived from a nonlinear least squares fit to the data, using the one-set-of-sites model. Errors are the standard deviations obtained from the fits. Phosphorylation was achieved in situ by the presence of 5 µM HPr, 120 nM enzyme I, and 2 mM PEP as described under "Experimental Procedures."

The Binding of Mannitol to Trypsin-treated EIImtl-- Mild trypsinolysis of EIImtl-containing membrane vesicles leads to the formation of two fragments of approximately equal molecular weight (5), an N-terminal fragment, the C domain of EIImtl, responsible for mannitol binding and transport across the membrane, and a C-terminal fragment consisting of the cytoplasmic A and B domains. At longer exposure times, trypsin fully degrades the A and B domains, but the C domain is protected from degradation, apparently as a result of its membrane environment. Removal of the A and B domains leaves the structural integrity and transport capability of the C domain intact, since upon complementation with a purified combination of the B and A domains, IIBAmtl, the activity of the system is restored (24). We have used this approach here to eliminate the A and B domains of EIImtl, enabling us to study the influence of domain interactions on mannitol binding. The results are shown in Fig. 6 and Table IV. Removal of the A and B domains results in an increase in Kd to values ranging from 200 to 400 nM, depending on temperature (Table IV). Delta Hoobs is around 57 kJ mol-1 and does not vary much with temperature compared with the wild type enzyme. The resulting value for Delta Cpoobs is -0.5 ± 0.2 kJ K-1 mol-1, much smaller than in the in the intact enzyme with the A and B domains present. All values determined for Delta Cpoobs in this study are listed in Table V.


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Fig. 6.   Integrated heats of injection for the titration of C domain of enzyme IImtl obtained from trypsinolysis (IICmtl) with mannitol in 50 mM potassium phosphate, pH 7.5, 2 mM EDTA, 1 mM DTT at 9.1 °C (bullet ), 19.9 °C (open circle ), and 24.9 °C (×). For clarity, not all data are shown. The solid lines represent the best fit of the one-set-of-sites model to the data. Inset, the variation of Delta Hoobs with temperature. The solid line is obtained from linear least squares analysis and has a slope of -0.5 ± 0.2 kJ K-1 mol-1.

                              
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Table IV
Mannitol binding by IICmtl, prepared from EIImtl by trypsinolysis
Parameters are derived from a non-linear least squares fit to the data, using the one-set-of-sites-model. Errors are the standard deviations obtained from the fits.

                              
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Table V
The observed heat capacity increment, Delta Cpoobs, and the theoretical number of residues involved in substrate binding, Rth
Delta Cpoobs was calculated from the data listed in Tables I-IV; Rth was calculated as described (15).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Validity of the One-set-of-sites Model for Binding of Mannitol to EIImtl-- To be able to quantitatively interpret the data obtained from isothermal titration calorimetry experiments, one must choose a mathematical model to analyze the data. Previous binding studies on substrate binding by EIImtl using equilibrium dialysis or filtration techniques indicated that there are two binding sites for mannitol on the EIImtl dimer, a high affinity site (HA) with a Kd of approximately 45-600 nM and a low affinity site (LA) with Kd values of 5-10 µM (25-27). Binding studies using flow dialysis resulted in values ranging from 45 to 212 nM for the HA binding site (11, 12), whereas the LA site could not be assessed with this technique. Based on this information, a binding model describing two possible binding sites with different affinities would seem to be the appropriate choice. However, attempts to fit the ITC data with such a model did not give sensible results and were, therefore, not further pursued. An explanation for this apparent discrepancy may be found in the values of the parameter c, the quotient of the concentration of sites in the calorimetric cell and the Kd,c should be between 1 and 1000 to be able to calculate the thermodynamic parameters reliably (21). Assuming a value of 5.2 µM for the Kd of the LA site (27), the value of the c parameter for this site is just above 1 in our experiments. For comparison, the values of c calculated from the data in Table I range from 32 to 210 and, therefore, it is not surprising that the observed dependence of the enthalpy on the molar ratio of mannitol and EIImtl is totally dominated by binding to the HA site. Further support for our interpretation comes from the fact that the observed dissociation constants for the HA site in phosphate and Tris buffer are in good agreement with values that were measured previously in inside-out membrane vesicles containing EIImtl, using other techniques.

The average value of the stoichiometry calculated from the data in Table I in the absence of detergent is 0.62 ± 0.06, somewhat higher than the expected value of 0.5 for one HA site per EIImtl dimer. This is probably due to inaccuracies in the concentration determination of EIImtl, possibly resulting from oxidation of a small fraction of the protein. The active site cysteine of the B domain, Cys-384, is the site most prone to oxidation, a process that results in the loss of phosphorylation activity but not substrate binding. Since our concentration determination is based on the former, this would result in an underestimation of the protein concentration and, therefore, an overestimation of the binding stoichiometry. It should be noted that introducing an erroneous concentration of binding sites into the data analysis does not alter the values obtained for the Kd and Delta Hoobs of the reaction (28).

Enthalpy-Entropy Compensation-- Although the range of reported enthalpies in the reactions investigated in this study is almost 80 kJ mol-1, the value of Delta Goobs varies only over a very narrow range (~6 kJ mol-1) for the entire data set. This phenomenon is termed enthalpy-entropy compensation and can be illustrated in a plot of Delta Hoobs versus Delta Soobs (Fig. 7). In the case of complete compensation, the expected slope of such a plot is the experimental temperature (29). For our data at 25 °C, the temperature at which we have the most complete data set with the largest range of Delta Hoobs and Delta Soobs, we find a slope of 303 ± 8 K, equal within error to the expected value of 298 K. This is additional evidence that the model we have chosen to represent our data is correct. It also implies that the temperature dependence of the enthalpy and entropy functions are dominated by solvent reorganization, or more specifically, by the removal of water from surfaces that become buried upon binding of the substrate.


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Fig. 7.   Enthalpy-entropy compensation for the binding of mannitol (mtl) and perseitol (ptl) to EIImtl, P2-EIImtl, and C domain of enzyme IImtl obtained from trypsinolysis (IICmtl), shown as a plot of Delta Hoobs (bullet ) and Delta Goobs (open circle ) versus Delta Soobs at 25 °C (298 K). Data were taken from Tables I-IV. The solid line is obtained from linear least squares analysis and has a slope of 303 ± 8 K. The dotted line represents the average value of Delta Goobs (-37.8 kJ mol).

Dependence of Ligand Binding on Temperature-- The heat capacity increment of the binding reaction, Delta Cpoobs, for the reaction of EIImtl and mannitol is -4.0 ± 0.3 kJ K-1 mol-1 in phosphate buffer. Qualitatively, this estimate can be interpreted as a conformational change of the transporter, resulting in removal of hydrophobic groups from the surrounding water (30). For a more quantitative analysis, care should be taken that the determination of Delta Cpoobs is not hampered by protonation effects, which can have a large influence on Delta Hoobs and Delta Cpoobs of binding (31). Simulations have shown that the largest deviations of Delta Cpoobs from the intrinsic value are expected at the pKa values of the complex and of the free protein. They are, at constant pH, a smooth function of the ionization enthalpy of the buffer and change in sign when the ionization enthalpies of protein and buffer are approximately equal. In the case of EIImtl, the most likely candidate to undergo a change in protonation state is a histidine (11), with an approximate ionization enthalpy of 30 kJ mol-1. We have determined Delta Cpoobs in phosphate and Tris buffers with ionization enthalpies that bracket the ionization enthalpy of histidine, and found that they differ by no more than 18% of the lowest value between the two. Therefore we conclude that the values we have determined are close to the intrinsic value for the Delta Cpoobs of the binding reaction of mannitol and EIImtl.

The values for Delta Cpoobs for the binding of mannitol and perseitol to EIImtl are large when compared with other binding processes. For example, the Delta Cpoobs for binding of serine to the serine receptor of bacterial chemotaxis is approximately -0.7 kJ K-1 mol-1 (32) and for binding of glucose to yeast hexokinase a value of -0.2 kJ K-1 mol-1 was observed (33). Larger values are observed when binding is coupled to folding and/or oligomerization; in the case of the lambda  Cro repressor, Delta Cpoobs is -6.4 kJ K-1 mol-1 (34), and for the binding of the glucocorticoid DNA binding domain to its DNA site, Delta Cpoobs is -4.2 kJ K-1 mol-1 (35). Although EIImtl is known to form dimers and, therefore, it could be argued that dimerization leads to the large value of Delta Cpoobs determined here, we do not believe that this is the case. The monomer-dimer equilibrium has been monitored by a number of techniques (3-9) and, in all of these cases, monomerization was observed only at concentrations in the nanomolar range and only in detergent. The concentrations we have employed in our study are much higher, and we are working with membrane vesicles with their natural complement of lipids. Thus, it seems fair to conclude that EIImtl is in its dimeric form in all experiments in this study. Consequently, the observed thermodynamic parameters must result from changes in the EIImtl dimer going from a free state to a substrate-bound state.

Spolar and Record (15) developed a method to determine the number of residues involved in a binding reaction from the value of Delta Cpoobs. Application of their analysis to our data yields estimates of approximately 50-60 residues for the binding of mannitol and 62 for the binding of perseitol (Table V). This analysis depends on an average value of the ratio of polar and non-polar surface area that is buried during the binding process determined from data for globular proteins. Therefore, in the absence of structural data, it can only give an indication of the actual number of residues involved, but it illustrates that a major conformational change takes place upon binding of either mannitol or perseitol to the EIImtl-dimer when the protein is embedded in the cytoplasmic membrane.

The Effect of Phosphorylation and Removal of the B Domain on Delta Cpoobs-- In the case of perseitol binding to phosphorylated EIImtl, Delta Cpoobs could not be determined accurately, but it is clear that the value is considerably smaller, although probably still rather large for the binding of a small molecule to a protein. The data do not permit a reliable calculation of the number of residues influenced by the binding, but we can qualitatively state that the number is much smaller when perseitol binds to phosphorylated versus unphosphorylated enzyme. Thus phosphorylation of the B domain influences the structural changes of the enzyme upon binding of perseitol to the C domain.

Removal of the A and B domain from EIImtl by trypsinolysis decreases Delta Cpoobs to -0.5 kJ K-1 mol-1. The theoretical value of the number of residue involved in the binding drops accordingly to a value of 9 residues, indicating that the conformational changes in the absence of the B and A domains involve considerably fewer residues than in the intact enzyme. Since it is known that there is no functional interaction between the A and C domains, we can attribute this difference solely to the absence of the B domain. Therefore, a very significant part of the structural changes occurring in the transporter upon binding of mannitol reside in the B domain, rather than the C domain.

Domain Interactions in EIImtl-- The results obtained after phosphorylation and trypsinolysis of the enzyme strongly suggest a conformational change upon binding of the substrate to the C domain that is propagated to the B domain. Other evidence for conformational coupling between the B and C domains of EIImtl has been obtained from mannitol binding and transport kinetics and their dependence on chemical changes and mutations at the active site of the B domain (10, 11). Our data support those conclusions and also suggest a mechanism for the coupling. The very large value of Delta Cpoobs and the enthalpy-entropy compensation behavior indicate that a new complementary surface is formed upon binding, either from folding of unstructured parts or docking of preexisting surfaces on the protein, but, in either case, resulting in the removal of solvent-exposed parts of the protein from the surrounding water. This is most likely to happen between the B and C domains of the enzyme, because the next step in the catalytic cycle of the enzyme is phosphorylation of the incoming mannitol by the B domain, a process requiring precise positioning of the phosphoryl group donor and acceptor. This would explain the decrease in Delta Cpoobs after removal of the B domain because one half of the complementary surface is no longer present. The fact that phosphorylation of the protein also leads to a large decrease in Delta Cpoobs can be explained in two different ways; either the surface is already formed upon phosphorylation so that binding of substrate is no longer required to generate complementary surfaces, or the newly formed surfaces cannot complement due to steric repulsion of the C7 moiety of the perseitol and the phosphoryl group attached to Cys-384 on the B domain. A possible way to discriminate between the two possibilities is to study the thermodynamics of mannitol binding in a series of mutants that mimic the phosphorylated state of the B domain; these studies are in progress.

    FOOTNOTES

* This work was supported by the Netherlands Foundation for the Life Sciences with grants from the Netherlands Organization for Scientific Research.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.

Dagger To whom correspondence should be addressed. Tel.: 31-50-3634321; Fax: 31-50-3634165; E-mail: got.orobillard{at}chem.rug.nl.

1 The abbreviations used are: PTS, phosphoenolpyruvate-dependent phosphotransferase system; DTT, dithiothreitol; EIImtl, enzyme IImtl; HA, high affinity; HPr, histidine-containing protein; LA, low affinity; mtl, mannitol; PEP, phosphoenolpyruvate; ITC, isothermal titration calorimetry; TPCK, tosylphenylalanyl chloromethyl ketone.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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