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

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. IIABManhas 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 β-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 vitroby 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 andtrans 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.

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)(2)(3)(4). IIA and IIB of certain transporters have regulatory activity in addition to their "energy-transducing" function. For instance, IIA Glc 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 ␤-glucoside transporter (IIBCA Bgl ) phosphorylates the antiterminator protein BglG and thereby inactivates it. This way, IIBCA Bgl 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 (IIA Glc ) (7), stable dimers (IIA-Man ) (19), or trimers (IIA Lac ) (20) of identical subunits. Similarly, the IIB units have different 3D structures but are monomeric (21)(22)(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 IICB Glc 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 IICBA Mtl (30,31) and between two inactive mutants of the paralogous transporters for Glc and for GlcNAc (IICB Glc and IICBA GlcNAc ) 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 IIAB Man 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 IIA Man domain contains a five-stranded ␤-sheet (strand order 21345) covered by helices on either face ((␤␣)4,␣␤). 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 ␤-sheet (strand order 3241567, 1-6 are parallel and 7 is antiparallel) covered by helices on both faces, as deduced from the IIB Lev subunit which is 47% identical to the IIB Man 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 ␤-strand and ␣-helix (23).
Only the A domain participates in the dimer interface. The monomer-monomer interaction occurs through the interlocked ␤-strands and an extensive contact area of 1700 Å 2 composed mainly of hydrophobic residues. This confers high stability, and the IIAB Man dimer can be dissociated only concomitant with complete denaturation (37). The B domain interacts with the transmembrane IIC Man ⅐IID Man complex of the mannose transporter. The IIAB Man ⅐IIC Man ⅐IID Man complex, which can be purified intact, has a stoichiometry closest to 2:1:2 (38 -40). The IIAB Man dimer can also be purified as a soluble protein. It has been shown previously (34) that the active site mutants of IIAB Man , 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 WA2127⌬HIC (manXYZ ptsHIcrr (42)) was transformed with derivatives of pJFL encoding wild-type and mutant IIAB Man (34). IIAB Man was overexpressed and purified as described (34). Enzyme I and HPr were purified, and membranes containing IIC Man ⅐IID Man were prepared as described (42,43).
GuHCl Unfolding and Renaturation of IIAB Man -Stock solution of purified wild-type and mutant IIAB Man 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 IIAB Man concentration (3-125 g/ml) and incubated for another 2 h at 4°C.
Assay for Phosphotransferase Activity-In vitro phosphorylation of [ 14 C]Glc was assayed by ion-exchange chromatography as described (34). 100 l of incubation mixture contained 0.5 g of enzyme I, 2. Assay for Protein Phosphorylation-The rate and the extent of protein phosphorylation was measured as described (45). The incubation mixture (50 mM NaP i , pH 7.4, 5 mM MgCl 2 , 2.5 mM NaF, 2.5 mM dithiothreitol) contained, per 250 l, 1.5 g of enzyme I, 2.5 g of HPr, and 85 g of IIAB Man . The phosphorylation reaction was started by adding to the incubation mixture at 24°C [ 33 P]PEP to a final concentration of 80 M. Aliquots of 40 l 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-l incubation mixtures contained 134 M [ 33 P]PEP, 0.15 g of enzyme I, 0.46 g of HPr, 10 g of IIAB Man , and 0.3 l of IIC Man ⅐IID Man -containing membranes.

Functional Interaction of Subunits in IIAB Man Dimers-
Wild-type IIAB Man , 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 IIAB Man . 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 bellshaped 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 IIAB Man 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 IIAB Man occurs as homodimer (no GuHCl) or whether only 11% of IIAB Man was in homodimers and the rest in heterodimers with an inactive subunit. The activity was linearly dependent upon the concentration of wildtype IIAB Man 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 IIAB Man 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 IIAB Man 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 homoand heterodimers) for the IIC Man ⅐IID Man complex. Competitive inhibition becomes more pronounced when the concentration of IIC Man ⅐IID Man 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).
With each experiment, a control with pure wild-type IIAB Man was carried along as a reference for 100% activity and as control of refolding yield. The activity recovered after rapid dilution of wild-type IIAB Man was 80 Ϯ 30% (Table I, column IIAB Man homodimer). The specific activity of heterodimers was calculated as follows. The activity contributed by IIAB Man wildtype 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 IIAB Man from experiment to experiment varies between 2500 min Ϫ1 and 1200 min Ϫ1 . The H10C⅐H175C heterodimer has a turnover of 370 min Ϫ1 . This is 37% of the activity of wild-type IIAB Man 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%.
Protein Phosphorylation-IIAB Man is phosphorylated with [ 33 P]PEP in the presence of enzyme I and HPr and is dephosphorylated in the presence of IIC Man ⅐IID Man 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 IIC Man ⅐IID Man and glucose, indicating that phosphorylation occurred at His-175. Phosphorylation of H10C is HPr-dependent but much slower than phosphorylation of wild-type IIAB Man (Fig. 5). His-175 must be phosphorylated by HPr directly. Contamination of H10C by IIAB Man , 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 wildtype IIAB Man or in H175C results in an increased stabilization of the IIAB Man dimer against dissociation by sodium dodecyl sulfate, and this effect is not reversed as a consequence of dephosphorylation by IIC Man ⅐IID Man and mannose (Fig. 4B). DISCUSSION IIAB Man consists of two domains, IIA and IIB, that sequentially transfer a phosphoryl group from the phosphoryl carrier protein HPr to the transported sugar. IIAB Man is a homodimer. The subunits are tightly linked through mutual exchange of ␤-strands between the ␤-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 IIAB Man 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 IIAB Man . 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 path-ways 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 IIAB Man , the interpretation has changed. The weak complementation was because of phosphoryl transfer between randomly colliding homodimers. IIAB Man 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 IIB Man ⅐IIC Man ⅐IID Man complex in either of two orientations (Fig. 1C). The cis orientation is presented in Fig. 1A.
A IIAB Man 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 surfaceexposed 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 genomes 2 (complete and in progress) of: Yersinia pestis, Actinobacillus actinomycetemcomitans, Entero-  Fig. 3, A-E and the control experiments with pure wild-type IIAB Man . 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 IIAB Man homodimers (in bold), and the concentrations of hetero-and homodimers in the mixtures derived from the binomial distribution (in italics). 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 (IICB Glc ⅐IIA Glc and IICBA GlcNAc ) are homologous, but whereas IICBA GlcNAc is a three-domain protein, IIA Glc and IICB Glc are independent subunits. IIA Glc 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)(6)(7)(8)(9)(10)(11)(12). These interactions with soluble and membrane-bound target proteins require that IIA Glc 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. FIG. 4. Phosphorylation and dimerization of IIAB Man . Purified IIAB Man was incubated with [ 33 P]PEP in the presence of enzyme I, HPr, and IIC Man ⅐IID Man for 10 min at 37°C. To one aliquot (ϩ) 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 IIAB Man . The polyacrylamide gel was autoradiographed (A) and then stained with Coomassie Blue (B). Note that the H10C mutation prevents the formation of IIAB Man dimers resistant to dissociation by sodium dodecyl sulfate.
FIG. 5. Time course of phosphorylation of IIAB Man . Purified wild-type IIAB Man (circles) and H10C (squares) was incubated with [ 33 P]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.