Mechanism of Phosphoryl Transfer in the Dimeric IIABMan Subunit of the Escherichia coli Mannose Transporter*

Regula Gutknecht, Karin Flükiger, Regina Lanz, and Bernhard ErniDagger

From the Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012, Bern, Switzerland

    ABSTRACT
Top
Abstract
Introduction
References

The mannose transporter of bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. It consists of the transmembrane IICMan·IIDMan complex and the cytoplasmic IIABMan subunit. IIABMan has two domains (IIA and IIB) that are linked by a 60-Å long alanine-proline-rich linker. IIABMan transfers phosphoryl groups from the phospho-histidine-containing phospho-carrier protein of the PTS to His-10 on IIA, hence to His-175 on IIB, and finally to the 6'-OH of the transported hexose. IIABMan occurs as a stable homodimer. The subunit contact is mediated by a swap of beta -strands and an extensive contact area between the IIA domains. The H10C and H175C single and the H10C/H175C double mutants were used to characterize the phosphoryl transfer between IIA to IIB. Subunits do not exchange between dimers under physiological conditions, but slow phosphoryl transfer can take place between subunits from different dimers. Heterodimers of different subunits were produced in vitro by GuHCl-induced unfolding and refolding of mixtures of two different homodimers. With respect to wild-type homodimers, the heterodimers have the following activities: wild-type·H10C, 50%; wild-type·H175C 45%; H10C·H175C, 37%; and wild-type·H10C/H175C (double mutant), 29%. Taken together, this indicates that both cis and trans pathways contribute to the maximal phosphotransferase activity of IIABMan. A phosphoryl group on a IIA domain can be transferred either to the IIB domain on the same or on the second subunit in the dimer, and interruption of one of the two pathways results in a reduction of the activity to 70-80% of the control.

    INTRODUCTION
Top
Abstract
Introduction
References

The carbohydrate transporters of the bacterial phosphotransferase system (enzymes II of the PTS)1 mediate uptake concomitant with phosphorylation of hexoses and hexitols. They consist of four functional units termed IIA, IIB, IIC, and IID that occur either as individual subunits in a protein complex or as independently folding domains of a multidomain protein. IIA and IIB sequentially transfer a phosphoryl group from the phosphoryl carrier protein HPr to the transported substrate. IIC and IID span the membrane and mediate substrate translocation. Substrate translocation is activated by the phosphorylation/dephosphorylation cycle of IIB (1-4). IIA and IIB of certain transporters have regulatory activity in addition to their "energy-transducing" function. For instance, IIAGlc of Escherichia coli, the gene product of crr, modulates the activities of adenylate cyclase (5, 6), glycerol kinase (7), and of the membrane permeases for lactose and maltose (8-12). The IIB domains of some PTS transporters regulate the activity of antiterminator and transcription activator proteins (13). In the absence of the cognate substrate, the IIB domain of the beta -glucoside transporter (IIBCABgl) phosphorylates the antiterminator protein BglG and thereby inactivates it. This way, IIBCABgl feedback inhibits its own expression in the absence of a transportable substrate (inducer) (14, 15).

The tertiary and quarternary structures of IIA units from different families of PTS transporters are completely unrelated. IIA occur as monomers (IIAGlc) (7), stable dimers (IIAMan) (19), or trimers (IIALac) (20) of identical subunits. Similarly, the IIB units have different 3D structures but are monomeric (21-23). The membrane-spanning IIC and IID subunits occur as oligomers, mostly dimers (24-28). The multidomain composition of the PTS transporters and their dimeric structure allows for various forms of interallelic and intergenic complementation. For instance, the coexpression of two mutated IICBGlc subunits of the glucose transporter with inactive B and C domains, respectively, resulted in complementation of transport activity (29). Complementation has also been observed between inactive mutants of IICBAMtl (30, 31) and between two inactive mutants of the paralogous transporters for Glc and for GlcNAc (IICBGlc and IICBAGlcNAc) of E. coli (32, 33). It is generally assumed that complementation in vitro is because of the formation of heterodimers between two different inactive subunits and not only to transient association of different inactive homodimers.

The E. coli IIABMan subunit is a homodimer (see Fig. 1A). Each monomer comprises two independently folding domains, the A domain (residues 1-133) and the B domain (residues 156-323) connected by a 23-residue long alanine-proline-rich linker (35, 36). The IIAMan domain contains a five-stranded beta -sheet (strand order 21345) covered by helices on either face ((beta alpha )4,alpha beta ). Four strands are parallel, and the fifth antiparallel strand which forms one edge of the sheet is swapped between the subunits in the dimer. His-10, which is phosphorylated during phosphoryl transfer from HPr to IIB, is located at the topological switchpoint of the fold. Its imidazole ring is hydrogen bonded to Asp-67, which acts as a general base increasing the nucleophilicity of the imidazole ring (19). The B domain contains a 180° twisted seven-stranded beta -sheet (strand order 3241567, 1-6 are parallel and 7 is antiparallel) covered by helices on both faces, as deduced from the IIBLev subunit which is 47% identical to the IIBMan domain. His-175, which accepts the phosphoryl group from His-10 and transfers it to the sugar, is located on an exposed loop between the first beta -strand and alpha -helix (23).

Only the A domain participates in the dimer interface. The monomer-monomer interaction occurs through the interlocked beta -strands and an extensive contact area of 1700 Å2 composed mainly of hydrophobic residues. This confers high stability, and the IIABMan dimer can be dissociated only concomitant with complete denaturation (37). The B domain interacts with the transmembrane IICMan·IIDMan complex of the mannose transporter. The IIABMan·IICMan·IIDMan complex, which can be purified intact, has a stoichiometry closest to 2:1:2 (38-40). The IIABMan dimer can also be purified as a soluble protein. Dissociated from the transmembrane IICMan·IIDMan complex, IIABMan has an elongated form. Ratios f/fo of 1.81 and 1.72 were calculated from the sedimentation coefficient (s20,w = 3.7 S) determined by analytical ultracentrifugation (37) and the diffusion coefficient (D = 4.73 10-7 cm2 sec-1) determined by dynamic light scattering, respectively. The axial ratio of >10:1 derived from f/fo (41) is compatible with a fully extended dimer (Fig. 1B) composed of the central A dimer (50 Å along the major axis), the two linkers (66 Å when fully elongated), and the two B domains (35 Å average diameter).


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Fig. 1.   Hypothetical model of the mannose transporter complex. A, HPr and the two monomers of the IIABMan complex are in different shades of gray. The orientation of the IIB (PDB code 1BLE) and HPr (1POH, (55)) in the complex with the IIA dimer (1PDO) are taken from the model proposed by Schauder et al. (23). The active site histidines H10, H175, and H15 are shown in ball and stick representation. IICMan and IIDMan span the membrane. The cartoon of IIABMan was produced using MOLSCRIPT (56). B, backbone representation of IIABMan with the alanine-proline-rich linkers in a fully extended conformation. C, schematic representation of cis and trans orientations of the IIA dimer relative to the IIB domains with monomers. Active site contacts are indicated in black.

It has been shown previously (34) that the active site mutants of IIABMan, H10C, and H175C, are completely inactive when assayed alone, but that approximately 3% of wild-type activity is recovered when the purified proteins are mixed in a 1:1 ratio. Here we show, that much higher activity is recovered when the purified mutants are mixed, completely unfolded with GuHCl, and then renatured. True heterodimers form only under these drastic conditions. Phosphoryl transfer between subunits within the dimer is very efficient, whereas transfer between different dimers is possible but inefficient.

    EXPERIMENTAL PROCEDURES

Bacterial Strains, Overproduction, and Purification of Proteins-- E. coli WA2127Delta HIC (manXYZ ptsHIcrr (42)) was transformed with derivatives of pJFL encoding wild-type and mutant IIABMan (34). IIABMan was overexpressed and purified as described (34). Enzyme I and HPr were purified, and membranes containing IICMan·IIDMan were prepared as described (42, 43).

GuHCl Unfolding and Renaturation of IIABMan-- Stock solution of purified wild-type and mutant IIABMan were adjusted to a protein concentration of 5 mg/ml. Volumes from the different stocks were mixed to achieve the desired molar ratios or molar fractions. The mixtures were then split in two aliquots. One aliquot was diluted with 8 M GuHCl to a final concentration of 4 M GuHCl (37), and to the other aliquot, the same volume of buffer A (10 mM MOPS, pH 7.0, 50 mM NaCl, 0.5 mM dithiothreitol) was added. Both samples were incubated for 2 h at room temperature. Both samples were then diluted 20-60-fold with buffer A to the desired a IIABMan concentration (3-125 µg/ml) and incubated for another 2 h at 4 °C.

Assay for Phosphotransferase Activity-- In vitro phosphorylation of [14C]Glc was assayed by ion-exchange chromatography as described (34). 100 µl of incubation mixture contained 0.5 µg of enzyme I, 2.8 µg of HPr, and 0.5 µl of crude membranes (~4 µg of protein) containing the IICMan·IIDMan complex. The final concentration of renatured IIABMan varied between 3 ng and 130 ng/100 µl of incubation mixture. The exact values are indicated in the figure legends. The specific activity of [14C]Glc was 1000 cpm/nmol.

Assay for Protein Phosphorylation-- The rate and the extent of protein phosphorylation was measured as described (45). The incubation mixture (50 mM NaPi, pH 7.4, 5 mM MgCl2, 2.5 mM NaF, 2.5 mM dithiothreitol) contained, per 250 µl, 1.5 µg of enzyme I, 2.5 µg of HPr, and 85 µg of IIABMan. The phosphorylation reaction was started by adding to the incubation mixture at 24 °C [33P]PEP to a final concentration of 80 µM. Aliquots of 40 µl were withdrawn at the indicated time points and diluted into 1 ml of 80% ammonium sulfate solution at 4 °C. The protein precipitates were collected on glass microfibre filters (GF/F, Whatman) under suction, washed, and counted in a liquid scintillation counter. The background counts because of enzyme I and HPr (less than 10%) were subtracted from the counts of the complete system. Phosphorylated proteins were analyzed on 17.5% polyacrylamide gels as described (21). 20-µl incubation mixtures contained 134 µM [33P]PEP, 0.15 µg of enzyme I, 0.46 µg of HPr, 10 µg of IIABMan, and 0.3 µl of IICMan·IIDMan-containing membranes.

    RESULTS

Functional Interaction of Subunits in IIABMan Dimers-- Wild-type IIABMan, H10C, H175C, and H10C/H175C double mutant were purified by phosphocellulose chromatography and gel filtration. A 1:1 mixture of purified H10C and H175C has about 5% of the specific activity of wild-type IIABMan. The activity increases nonlinearly at low concentration, and the concentration activity profile does not change after 24 h of preincubation (Fig. 2). These results suggest that the activity is because of transient association between two different inactive homodimers (second order reaction) and that monomers do not exchange to form heterodimers. However, when mixtures of H10C and H175C were denatured in GuHCl and then refolded by rapid dilution, a 20-fold higher specific phosphotransferase activity was obtained (Fig. 3A). When H10C and H175C were mixed in different proportions, the activity profile was bell-shaped with a maximum at a 1:1 molar ratio (Fig. 3B), as expected for a binomial distribution of active heterodimers and inactive homodimers. The activities of heterodimers between wild-type and mutated subunits was characterized in the following experiments. Constant amounts of wild-type IIABMan were mixed with increasing amounts of either H10C or H175C. One-half of the mixture was denatured with GuHCl and then renatured by dilution, the other was diluted only. The phosphotransferase activity remained approximately constant at all concentrations of H10C and H175C (Fig. 3C) independently of whether 100% of wild-type IIABMan occurs as homodimer (no GuHCl) or whether only 11% of IIABMan was in homodimers and the rest in heterodimers with an inactive subunit. The activity was linearly dependent upon the concentration of wild-type IIABMan when wild-type and H10C or H175C were mixed in different molar ratios, denatured, and then renatured (Fig. 3D). This suggests that the presence of a subunit with only one inactive domain in a heterodimer has no effect on the overall phosphotransferase activity of the wild-type subunit. Mixtures between wild-type IIABMan and an excess of the H10C/H175C double mutant were prepared to characterize the phosphoryl transfer between A and B domains on the same subunit. The concentration of wild-type IIABMan was kept constant, and the concentration of the double mutant increased to a maximum of 16:1 (Fig. 3, E and F). At a concentration ratio of 16:1, when only 6% of the wild-type protein is in homodimers and 94% in heterodimers with the double mutant, the activity is still 60% of the control and identical to the activity of the nondenatured mixture. The 40% decrease of activity is because of competition of the excess of inactive homodimers (8-fold over active homo- and heterodimers) for the IICMan·IIDMan complex. Competitive inhibition becomes more pronounced when the concentration of IICMan·IIDMan is rate-limiting. Under these conditions, the phosphotransferase activity is reduced to 50% when the concentration of wild-type homodimer plus heterodimer equals the concentration of the H10C/H175C homodimer (Fig. 3F).


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Fig. 2.   Phosphotransferase activity of mixtures of H10C and H175C. Purified H10C and H175C were mixed, preincubated for 30 min (squares) and 24 h (triangles), and then assayed. Wild-type IIABMan was preincubated for 30 min (circles) and then assayed.


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Fig. 3.   Effect of GuHCl unfolding/renaturation upon phosphotransferase activity of IIABMan mixtures. Open symbols, denatured/renatured mixtures; closed symbols, native mixtures. A, effect of renaturation/denaturation on the activity of a 1:1 mixture of H10C and H175C (42 ng of IIABMan/µl added). B, complementation between H10C and H175C at the indicated molar fractions (125 ng of IIABMan per assay point). C, noncomplementation between wild-type IIABMan and H10C (circles) and H175C (squares) in the presence of an excess of the mutant over wild-type IIABMan (3 ng of wild-type IIABMan per assay point at all molar ratios). D, noncomplementation between wild-type IIABMan and H10C (circles) and H175C (squares) at the indicated molar fractions (6.2 ng of IIABMan per assay point). E, negative dominant effect of the H10C/H175C double mutant over wild-type IIABMan in the presence of an excess of IICMan·IIDMan (7 ng of wild-type IIABMan per assay point at all molar ratios). F, same as panel E but with a limiting concentration of IICMan·IIDMan. Means and S.D. are of three experiments.

With each experiment, a control with pure wild-type IIABMan was carried along as a reference for 100% activity and as control of refolding yield. The activity recovered after rapid dilution of wild-type IIABMan was 80 ± 30% (Table I, column IIABMan homodimer). The specific activity of heterodimers was calculated as follows. The activity contributed by IIABMan wild-type homodimers was subtracted from the total phosphotransferase activity of a mixture of all dimers. The resulting difference was then divided by the concentration of heterodimers in the mixture. The concentrations of homo- and heterodimers were calculated from the binomial distribution. The specific activities of the different dimers are summarized in Table I. The turn-over number of wild-type IIABMan from experiment to experiment varies between 2500 min-1 and 1200 min-1. The H10C·H175C heterodimer has a turnover of 370 min-1. This is 37% of the activity of wild-type IIABMan measured under the same conditions. The turn-over numbers of heterodimers between a wild-type subunit and either H10C or H175C are 50 and 45% of wild-type homodimer, and the turnover-number of a heterodimer between a wild-type subunit and a H10C/H175C double mutant is 30%.

                              
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Table I
Phosphotransferase activity of IIABMan dimers
The specific activities were calculated from Fig. 3, A-E and the control experiments with pure wild-type IIABMan. The specific activities (in bold) of the homo- and heterodimers were calculated from the measured activity of the mixtures of dimers, the measured specific activity of pure wild-type IIABMan homodimers (in bold), and the concentrations of hetero- and homodimers in the mixtures derived from the binomial distribution (in italics).

Protein Phosphorylation-- IIABMan is phosphorylated with [33P]PEP in the presence of enzyme I and HPr and is dephosphorylated in the presence of IICMan·IIDMan and glucose (Fig. 4A). The H175C mutant is stably phosphorylated at His-10 but cannot be dephosphorylated because His-175 is missing. The H10C mutant is weakly phosphorylated although His-10 is missing. It is dephosphorylated in the presence of IICMan·IIDMan and glucose, indicating that phosphorylation occurred at His-175. Phosphorylation of H10C is HPr-dependent but much slower than phosphorylation of wild-type IIABMan (Fig. 5). His-175 must be phosphorylated by HPr directly. Contamination of H10C by IIABMan, which could complement the IIA function, can be excluded because H10C was isolated from an E. coli strain with a chromosomal deletion of the manXYZ operon. It is likely, that phosphorylation of IIB is a consequence of high local concentration of HPr which binds to mutated IIA and then nonspecifically delivers the phosphoryl group to a nearby His-175. Phosphorylation at His-10, whether in wild-type IIABMan or in H175C results in an increased stabilization of the IIABMan dimer against dissociation by sodium dodecyl sulfate, and this effect is not reversed as a consequence of dephosphorylation by IICMan·IIDMan and mannose (Fig. 4B).


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Fig. 4.   Phosphorylation and dimerization of IIABMan. Purified IIABMan was incubated with [33P]PEP in the presence of enzyme I, HPr, and IICMan·IIDMan for 10 min at 37 °C. To one aliquot (+) a molar excess of Glc was added to dephosphorylate the PTS proteins. Note that the H10C mutant is weakly phosphorylated at His-175 and dephosphorylated by glucose, and that H175C is strongly phosphorylated but not dephosphorylated. The double mutant H10C/H175C is not phosphorylated, a mixture of the H10C and H175C behaves like wild-type IIABMan. The polyacrylamide gel was autoradiographed (A) and then stained with Coomassie Blue (B). Note that the H10C mutation prevents the formation of IIABMan dimers resistant to dissociation by sodium dodecyl sulfate.


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Fig. 5.   Time course of phosphorylation of IIABMan. Purified wild-type IIABMan (circles) and H10C (squares) was incubated with [33P]PEP in the presence of catalytic amounts of enzyme I with HPr (open symbols) and without HPr (closed symbols). The reaction was stopped at the indicated time points by ammonium sulfate precipitation. Protein precipitates were collected on filters and counted.


    DISCUSSION

IIABMan consists of two domains, IIA and IIB, that sequentially transfer a phosphoryl group from the phosphoryl carrier protein HPr to the transported sugar. IIABMan is a homodimer. The subunits are tightly linked through mutual exchange of beta -strands between the beta -sheets of IIA (19). The B domains, in contrast, neither interact with each other nor strongly interact with the IIA domains to which they are, however, covalently linked via 60-Å long alanine-proline-rich linker (Fig. 1, A and B). Phosphoryl groups can be transferred from IIA to IIB on the same subunit (cis), on different subunits (trans), or both. Our results indicate that cis and trans pathways are of comparable efficiency. Wild-type IIABMan with four sites and four pathways (two cis and two trans) per dimer has the highest specific activity. The heterodimer between wild-type and H10C or H175C with three active sites and only two pathways (one cis and one trans) has 50% specific activity. The active monomer in this heterodimer retains its full activity. Heterodimers with only one functional A and one functional B domain and only one pathway (cis or trans) retain between 30 and 40% activity. Taken together, this indicates that both cis and trans pathways contribute to the maximal phosphotransferase activity of IIABMan. A phosphoryl group on a IIA domain can be transferred either to the IIB domain on the same or on the second subunit in the dimer, and interruption of one of the two pathways results in a reduction of the activity by 20% to 30% of the control.

The results confirm our previous observation of interallelic complementation (34) and similar observations by others (26, 31, 46, 47). But in the case of IIABMan, the interpretation has changed. The weak complementation was because of phosphoryl transfer between randomly colliding homodimers. IIABMan monomers do not exchange, as evident from the structure of the IIA dimer (19). However, the long linker (Fig. 1B) allows sterically unconstrained interaction between IIA and IIB domains on different dimers. The linker allows the IIA dimer to dock on the IIBMan·IICMan·IIDMan complex in either of two orientations (Fig. 1C). The cis orientation is presented in Fig. 1A.

A IIABMan mutant with His-86 on the IIA domain replaced by Asn was described to have the same properties as H175C mutant with an inactive IIB domain (34, 36). However, the x-ray structure of IIA showed that His-86 is in a surface-exposed loop and far from the active site. In addition, His-86 is not conserved in any of the homologous proteins (see below). Both observations make His-86 an unlikely target for mutations with a strong phenotype. Resequencing showed the supposed H86N mutation to contain the H175C mutation. We conclude that the H86N mutant is neutral and that vectors must have been exchanged by mistake.

Bacillus subtilis, Klebsiella pneumoniae, Vibrio furnissii,and Lactobacillus casei express transporters homologous to the mannose transporter of E. coli except that IIA and IIB are expressed as separate proteins subunits and not as two domains connected by an alanine-proline-rich linker (48-51). Using the Basic Local Alignment Search Tool (BLAST) program, IIAB homologs with alanine-proline-rich or Q-linkers (52, 53) were found in bacterial genomes2 (complete and in progress) of: Yersinia pestis, Actinobacillus actinomycetemcomitans, Enterococcus faecalis, Clostridium acetobutylicum, Streptococcus pneumoniae, and Streptococcus pyogenes. Why two forms of IIAB units and what function if any does the linker have? All things being equal, binding of the IIABMan dimer to the IICMan·IIDMan complex must be much stronger than binding of a monomeric IIB subunit because the dimer forms two contacts per molecule, whereas a IIB monomer forms only one (54). Although not covalent in the chemical sense, binding might become very strong, and IIAB remain membrane-bound for most of the time. Untying of IIB from the IIC·IID complex is necessary whenever IIB has a regulatory function and must diffuse to other targets. For example, monomeric IIBLev of B. subtilis is not only a subunit of the fructose transport complex, but it also can phosphorylate and thereby inactivate the transcriptional activator LevR (16-18). An analogous situation is observed in E. coli. The transporter for Glc and GlcNAc (IICBGlc·IIAGlc and IICBAGlcNAc) are homologous, but whereas IICBAGlcNAc is a three-domain protein, IIAGlc and IICBGlc are independent subunits. IIAGlc plays a pivotal role in regulation of catabolite repression and inducer inclusion, and it has been shown to interact with glycerol kinase, the transporters for lactose and maltose, and adenylate cyclase (5-12). These interactions with soluble and membrane-bound target proteins require that IIAGlc can freely diffuse through the cell.

The structural stability of the IIAB dimers and their mechanism of phosphoryl transfer might be unique among the different families of dimeric PTS transporters. Nevertheless, it indicates that interactions between different subunits within a dimer (first order reaction) as well as interactions between different dimers (second order reaction) have to be taken into consideration when weak interallelic complementation is observed. The ease with which stable heterodimers can be generated by reversible unfolding will facilitate the characterization by fluorescence energy transfer of domain motions that might occur during phosphorylation and transport of mannose.

    ACKNOWLEDGEMENTS

We thank S. Mukhija (ARPIDA AG, Münchenstein) for the gift of [33P]PEP, S. D. Snyder (Protein Solutions Inc., Charlottesville) for determining the diffusion coefficient by dynamic light scattering, and S. Schauder for the help with preparing Fig. 1.

    FOOTNOTES

* This study was supported by Grant 31-45838.95 from the Swiss National Science Foundation and by contributions from the Ciba-Geigy Jubiläumsstiftung, Basel, Switzerland.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.

The atomic coordinates and structure factors (codes 1BLE, 1POH, and 1PDO) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

Dagger To whom correspondence should be addressed. Tel.: ++41 (0)31/631 43 46; Fax: ++41 (0)31/631 48 87; E-mail: erni{at}ibc.unibe.ch.

2 http://www.ncbi.nlm.nih.gov/BLAST/unfinishedgenome.html.

    ABBREVIATIONS

The abbreviations used are: PTS, phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (EC 2.7.1.69); IIABMan, hydrophilic subunit of the mannose transporter; IICMan and IIDMan, transmembrane subunits of the mannose transporter; HPr, histidine-containing phospho carrier protein of the PTS; PEP, phosphoenolpyruvate; GuHCl, guanidinium hydrochloride; MOPS, 4-morpholinepropanesulfonic acid.

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Abstract
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