The Glucose Transporter of the Escherichia coliPhosphotransferase System

The glucose transporter of the bacterial phosphotransferase system (PTS) consists of a hydrophilic (IIAGlc) and a transmembrane subunit (IICBGlc). IICBGlc has two domains (C and B), which are linked by a highly invariant sequence. Transport of glucose by IIC and phosphorylation by IIB are tightly coupled processes. Three motifs that are strongly conserved in 12 homologous PTS transporters, namely two invariant arginines (Arg-424 and Arg-426) adjacent to the phosphorylation site (Cys-421), the invariant interdomain sequence KTPGRED, and two conserved histidines (His-211 and His-212) in the IIC domain were mutated and the mutant proteins characterized in vivo and in vitro for transport and phosphorylation activity. Replacement of the strongly β-turn favoring residues Thr and Gly of the linker by α-helix favoring Ala results in strong reduction of activity, whereas the substitutions of the other residues have only minor effects. The R424K and R426K mutants can be phosphorylated by IIAGlc but can no longer donate the phosphoryl group to glucose. The H211Q and H212Q mutants continue to phosphorylate glucose at a reduced rate but H212Q can no longer transport glucose. Mixtures of purified R424K/H212Q and R426K/H212Q have 10% of wild-type phosphorylation activity and when coexpressed inEscherichia coli support glucose transport.

The glucose transporter of the bacterial phosphotransferase system (PTS) consists of a hydrophilic (IIA-Glc ) and a transmembrane subunit (IICB Glc ). IICB Glc has two domains (C and B), which are linked by a highly invariant sequence. Transport of glucose by IIC and phosphorylation by IIB are tightly coupled processes. Three motifs that are strongly conserved in 12 homologous PTS transporters, namely two invariant arginines (Arg-424 and Arg-426) adjacent to the phosphorylation site (Cys-421), the invariant interdomain sequence KT-PGRED, and two conserved histidines (His-211 and His-212) in the IIC domain were mutated and the mutant proteins characterized in vivo and in vitro for transport and phosphorylation activity. Replacement of the strongly ␤-turn favoring residues Thr and Gly of the linker by ␣-helix favoring Ala results in strong reduction of activity, whereas the substitutions of the other residues have only minor effects. The R424K and R426K mutants can be phosphorylated by IIA Glc but can no longer donate the phosphoryl group to glucose. The H211Q and H212Q mutants continue to phosphorylate glucose at a reduced rate but H212Q can no longer transport glucose. Mixtures of purified R424K/H212Q and R426K/H212Q have 10% of wild-type phosphorylation activity and when coexpressed in Escherichia coli support glucose transport.
Escherichia coli has two transporters for glucose (1,2). They act by a mechanism that couples translocation with phosphorylation of the substrate. Both transporters are components of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). 1 The IIA Glc -IICB Glc complex is specific for glucose, the IIAB Man -IIC Man -IID Man complex has a broad substrate specificity for Glc, Man, GlcNAc, and other derivatives of Glc altered at the C2 carbon. The PTS comprises two cytosolic proteins, enzyme I and HPr, which sequentially transfer phosphoryl groups from PEP to the different carbohydrate-specific transporters (enzymes II). Number and substrate specificity of the transporters varies for different bacterial species (for re-view see Refs. [3][4][5]. They have been grouped into four families (6) based on amino acid sequence comparisons. IICB Glc belongs to the glucose family presently comprising 25 members (Protein Domain Data base; http://protein.toulouse.inra.fr/prodom/ prodom.html), the IIAB Man -IIC Man -IID Man belongs to the mannose family comprising 6 members (7-12).
All PTS transporters consist of three functional units (IIA, IIB, IIC), which occur either as protein subunits or domains of a multidomain polypeptide (13). The glucose transporter consists of two subunits IIA Glc and IICB Glc . IIA Glc is a 18-kDa hydrophilic protein that is phosphorylated at His-90 (14). The 51-kDa IICB Glc subunit consists of two domains (15). The hydrophobic domain (IIC, residues 1-ϳ380) spans the membrane eight times and contains the glucose binding site (16). The hydrophilic domain (IIB, residues ϳ380 -477) is phosphorylated at Cys-421 (17). Phosphoryl groups are transferred from HPr via His-90 of IIA Glc to Cys-421 and hence to glucose (18). The IIC and IIB domain are connected by an heptapeptide sequence that is highly conserved in those two-and threedomain transporters of the glucose family that have the domain order CB(A). A chimeric protein consisting of the IIC Glc domain of the glucose transporter and the IIBA GlcNAc domain of the GlcNAc transporter is active and glucose-specific (19). The IIC and IIB domains of IICB Glc can be expressed as separate polypeptides. The purified subclonal domains retain 2% of wildtype phosphotransferase activity when they are combined in vitro (20).
The three-dimensional structure of the IIB Glc domain, its interaction with the IIA Glc subunit and the structural consequences of its phosphorylation at Cys-421 have been analyzed by heteronuclear NMR spectroscopy (21,22). A model of the transmembrane topology of the IIC domain has been derived from protein fusion studies and further confirmed by linker insertion mutagenesis (16). 2 Point mutants of IICB Glc have been selected that facilitate Glc transport uncoupled of phosphorylation (23) and that retain glucose phosphorylation activity but have a strongly reduced translocation activity (24). All of these mutations are located in the IIC domain, and none was found so far in the IIB domain.
When the amino acid sequences of the transporters belonging to the glucose family are compared, several regions of strong amino acid similarity can be discerned (Fig. 1). One includes the active site Cys-421 which is phosphorylated by IIA Glc and donates the phosphoryl group to the transported glucose. Besides Cys-421, Arg-424, and Arg-426 are also invariant. They are the only invariant arginines in the transporters of the glucose family. Arginines are frequently found in phosphate-binding and phosphate-catalytic sites where they can stabilize phosphate through hydrogen bonding and electro-static interactions with their guanidino group (25). Here we characterize by Arg to Lys substitutions the functions of the two invariant arginines in the IIB domain. Surprisingly, the KTPGRED motif, the putative linker between the IIB and IIC domain, is also invariant. Although interdomain regions, linkers and hinges are enriched for residues such as alanines and prolines, glycines or glutamines (26 -28) their amino acid sequence is usually not conserved. Here we address the question of how important the residues of this heptapeptide are for the function of IICB Glc . A third conserved region surrounds the invariant His-212, which has once been proposed to be a potential phosphorylation site (28). Although it has been shown convincingly that a cysteine of the B domain and not the C domain is phosphorylated (17,29,30), the possible function of the highly conserved His-212 in the enzyme II components of the glucose family has not yet been elucidated. Here we show that this residue is essential for transport but not for phosphorylation activity.

EXPERIMENTAL PROCEDURES
Bacterial Strains, Plasmids, and Growth Media-E. coli K12 ZSC112L⌬G (glk manZ ⌬ptsG:Cm) was used as host for all experiments (20). Plasmid pTSGH11 encodes under the control of Ptac a IICB Glc with a carboxyl-terminal hexahistidine tag. pTSGH11 was constructed by ligation of the SacII-HindIII vector fragment of pTSG11 (20) and the SacII-HindIII insert fragment of pQEGH12 (31). pABG421 encodes the C421S mutant kanamycin resistance and the P15A origin of replication (20). Cells were grown at 37°C in LB medium containing appropriate antibiotics. XL1-blue (Stratagene) was used for cloning and plasmid amplification.
In Vivo Complementation Assays-ZSC112L⌬G was doubly transformed with pABG421 and pTSGH11 derivatives encoding the different IICB Glc mutants and streaked on McConkey plates containing ampicillin, kanamycin, and 0.4% Glc.
Overproduction and Purification of Proteins-E. coli ZSC112L⌬G was transformed with derivatives of pTSGH11 encoding wild-type and mutant IICB Glc . IICB Glc was overexpressed and purified by metal chelate affinity chromatography as described (20). Enzyme I, HPr, and IIA Glc containing cell extracts were prepared, and the proteins were purified as described (35).
In Vivo Transport and in Vitro Phosphotransferase Assays-Uptake of [ 14 C]␣MG by bacteria was assayed as described (24). Cells were grown to A 600 ϭ 0.5 in 250 ml of M9 mineral medium supplemented with 1% glycerol and 20 M isopropyl-1-thio-␤-D-galactopyranoside, collected by centrifugation, and resuspended in M9 medium without supplement. 0.33 ml of concentrated cell suspension were diluted with 0.77 ml of M9 medium and aerated for 10 min at 22°C. Uptake was started by the addition of 12 l of 10 mM [ 14 C]␣MG (6000 cpm/nmol). Aliquots of 100 l were withdrawn, diluted into 8 ml of ice-cold M9 containing 0.4 mM ␣MG, filtered through GF (Whatman) glass fiber filters under suction, and washed with 20 ml of ice-cold 0.15 M NaCl. The filters were dried and counted. The dry weight was determined from 0.2 ml of the concentrated cell suspension. In vitro phosphorylation of [ 14 C]Glc was assayed by ion-exchange chromatography as described (1). 100 l of incubation mixture contained 4 l of cell extract as source of enzyme I, HPr, and IIA Glc , 1 mM PEP, 1 mM [ 14 C]Glc (1000 dpm/nmol), 0.1 mg of E. coli phospholipid suspension (Sigma). The phosphorylated proteins were analyzed on 17.5% polyacrylamide gels as described (36). 20 l of incubation mixtures contained 75 M [ 32 P]PEP (1200 dpm/nmol), 0.7 g of enzyme I, 1.4 g of HPr, 0.3 g of IIA Glc , 0.1 mg of E. coli phospholipids, and 5 g of purified IICB Glc and where indicated 3 mM Glc. [ 32 P]PEP was prepared as described (37).

Alanine-scanning Mutagenesis of the Interdomain Linker-
The amino acid sequence KTPGRED between the IIC and IIB domains is the most highly conserved region in the transporters belonging to the glucose family of the PTS (Fig. 1). Six of these seven residues were replaced one at the time by alanine. Pro-384 was replaced with glycine because several attempts to replace it with Ala by gapped duplex and polymerase chain reaction-based mutagenesis methods failed. The reason for this failure is not known. Mutant proteins were expressed in strain ZSC112⌬G, which carries a chromosomal deletion of ptsG. Six transformants produced red colonies on McConkey glucose indicator plates, and only the G385A mutant produced yellow colonies with a small red center. All the IICB Glc mutants could be overepressed in the same amount as wild-type protein and purified by Ni 2ϩ chelate affinity chromatography (results not shown). The purified G385A protein displayed only 0.8% of wild-type activity, T383A had 10% of wild-type phosphotransferase activity, whereas the other mutants had between 40 and 120% of wild-type activity (Fig. 2). The very low activity of the G385A mutation was not due to protein degradation, since the protein could be purified in the same amount as the other IICB Glc mutants.
Function of the Invariant Arginines and Histidines-The IICB Glc mutants H211Q, H212Q, R424K, and R426K were expressed in strain ZSC112⌬G. The H212Q, R424K, and R426K mutants produced yellow colonies on McConkey glucose indicator plates indicating that they were unable to transport and phosphorylate glucose in amounts sufficient to support fermentation of glucose (results not shown). The H211Q mutant produced yellow colonies with a red center. Uptake of ␣MG by whole cells expressing H211Q was reduced to less than 10% of the wild-type control, whereas the H212Q, R424K, and R426K mutants had no transport activity (Fig. 3).
The four mutant proteins could be overexpressed in the usual amounts and purified by Ni 2ϩ chelate affinity chromatography. H211Q, H212Q, R424K, and R426K could be phosphorylated in the presence of [ 32 P]PEP, enzyme I, HPr, and IIA Glc , indicating that none of these residues is required for phosphoryltransfer from IIA Glc to Cys-421 on the IIB domain (Fig. 4). Upon addition of glucose to the incubation mixture the H211Q and H212Q mutants are dephosphorylated like the wild-type protein indicating that phosphoryl transfer between Cys-421 of IIB and glucose is still possible. In contrast, the R424K and R426K mutants remain phosphorylated in the presence of glucose indicating that both residues are required for the phosphoryl transfer from Cys-421 to glucose. The C421S mutant, which lacks the active site cysteine, is not phosphorylated (34). The glucose phosphotransferase activities of the purified proteins were determined in the standard sugar phosphotransferase assay (Fig. 5A). The H211Q mutant had almost the same specific activity as the wild-type control, the H212Q mutant retained 15% phosphotransferase activity, whereas the R424K and R426K mutants were inactive. Note, that the vectorial transport (uptake) and the nonvectorial phosphorylation activities are differentially affected by the His substitutions in the C domain, whereas both activities are completely inhibited by the Arg substitutions in the B domain.
Since IICB Glc is a homodimeric protein (18), interallelic complementation between different mutant subunits is possible. Cells expressing the H211Q, H212Q, R424K, and R426K mutant were transformed with a second plasmid encoding the inactive C421S mutant of IICB Glc and analyzed for glucose fermentation on McConkey plates. R424K/C421S and R426K/ C421S formed yellow colonies. However, the H212Q/C421S double transformants formed red colonies (results not shown) and showed transport activity in the whole cell uptake assay (Fig. 3). Interallelic complementation could also be observed with purified proteins in vitro. Mutant proteins were mixed in different ratios, whereas the total IICB Glc concentration was kept constant. The H212Q/R424K and H212Q/R426K combinations showed complementation (Fig. 5B). The heterodimers had approximately 10% of wild-type PTS activity when the mutant ratio was 1:1 (binomial distribution of heterodimers and homodimers). The mixture of purified R424K and R426K remained inactive. Mixtures of H211Q with inactive H212Q or R424K showed no complementation. The activity, which is 15% of wild-type activity for pure H211Q, decreased in proportion to the decreasing concentration of H211Q. H211Q/C421S and H211Q/R426K mixtures showed only weak complementation of phosphotransferase activity (Fig. 5C).

DISCUSSION
Point mutations in three strongly conserved regions of the IICB Glc subunit of the glucose transporter were characterized with respect to their effect on protein phosphorylation, nonvectorial glucose phosphorylation, and transport activity. The first region of interest is the KTPGRED linker between the IIC and the IIB domains, which is predicted to assume a loop structure (38,39). The T383A and G385A substitutions reduced the activity to less than 10% of the control. Thr and Gly favor ␤-turn structures, whereas Ala favors ␣-helices. Substitution of these ␤-turn residues at the center of the hinge by Ala could lead to a stiffening of the linker with concomitant reduced domain mobility. The G385A mutation is less active than the two domains expressed as completely separated subunits (20) or the transposition of the B domain from the carboxyl-terminal to the amino-terminal end of the IIC domain. IIBC Glc with circularly permuted domains has 40 -70% activity provided the two domains are linked via a flexible Ala Pro rich hinge peptide. 3 Pro is another residue that strongly favors ␤-turn structure. The P384G substitution reduced activity to 40%. Pro-384 could not be substituted by Ala for unknown reasons. It is likely that the KTPGRED linker acts as a hinge of precise but restricted mobility. In this respect it differs from the Ala Pro rich linkers, which serve as universal joints in many unrelated multidomain proteins including PTS proteins (26,28,40). The KTPGRED sequence is highly conserved only in the two domain proteins with the domain order CB but is missing in the two-subunit complexes of the glucose family and in the transporters that have the circularly permuted domain order BC.
His-211 and His-212 are conserved residues in the IIC domain, and Arg-424 and Arg-426 are two invariant residues immediately adjacent to the phosphorylation site of the IIB domain (Fig. 6). His-211 is not essential for any of the known IICB Glc functions. In contrast His-212, Arg-424, and Arg-426 are essential for vectorial transport of glucose. Phosphoryl transfer from Cys-421 to glucose is completely blocked in the arginine mutants and slow in the H212Q mutant. A mutant with a similar phenotype as R424K and R426K was also found in the IIB domain of the IIAB Man subunit of the structurally 3 R. Gutknecht and B. Erni, unpublished results. unrelated mannose transporter (41). IIAB Man R172Q can be phosphorlyated at His-175 but cannot donate the phosphoryl group to glucose. It is not clear what function Arg-424 and Arg-426 serve. In view of their proximity to the active site cysteine (Fig. 6) they could stabilize the phosphate in the bonded ground state or in the transition state during transfer from Cys-421 to Glc. Phosphotyrosine phosphatases, the second group of proteins that form phosphocysteine intermediates, provide strong evidence for such an interaction (42). Their active sites contain a cysteine and an arginine, separated by five residues. X-ray structures of these proteins indicate that the invariant arginine stabilizes the transition state with three hydrogen bonds between the guanidino group and two phosphate oxygens (43). However, since neither the IIB domains of the mannitol transporter nor the IIB domain of the cellobiose transporter have invariant arginines near the active site cysteine (44), alternative functions like stabilization of the interaction between the IIB and IIC domains during transport and phosphorylation of glucose should also be considered. In this case it is likely that second site suppressor mutations in the IIC domain can be found. The tight phenotype of the two arginine mutations will facilitate the selection of these mutants.
Transport activity is partially restored when H212Q and C421S are expressed together, and phosphotransferase activity is restored when purified H212Q is mixed with either R424K or R426K. This indicates that IICB Glc subunits cooperate in phosphoryltransfer from Cys-421 of one subunit to glucose bound to an intact IIC domain of the second subunit. Since IICB Glc is a dimer (18) this phosphoryl transfer most likely occurs between domains on different subunits within the dimer. However, in-terdimer phosphoryl transfer cannot be excluded, because molecular subunit exchange between purified dimers has not been demonstrated for IICB Glc . Indeed, as determined by fluorescence energy transfer, 4 intersubunit exchange is very slow in the IIAB Man dimer of the mannose transporter where the subunits are interwined through a swap of strands between the ␤-sheets of each subunit (45).
His-195 and Gly-196 are two invariant residues in the members of the mannitol family of PTS transporters. Like His-212 of IICB Glc , His-195 of IICBA Mtl was once considered as a potential phosphorylation site (46). His-212 and His-195 are located in the putative large cytoplasmic loop of their IIC domains (16,47). His-195 and Gly-196 of IICBA Mtl have been mutagenized and characterized (48 -50). Like H212Q of IICB-Glc , H195A and H195R of IICBA Mtl were inactive but surprisingly could not be complemented with a C384S mutant (equivalent of the C421S mutant of IICB Glc ) (48). The H195N mutant on the other hand was active (48). This indicates that His-195 of IICBA Mtl and His-212 of IICB Glc are functionally nonequivalent despite their apparently similar position in the primary structure. In contrast, the G196D mutant, which had less than 0.1% of wild-type activity, could be complemented with the C384S mutation, indicating that the IIB domain can transfer its phosphoryl group to mannitol bound to the IIC domain (49). Complementation between IIC and IIB as well as between IIB and IIA domains therefore appears to be a general property of the dimeric PTS transporters. Although complementation between sites on different domains is most probable (51), noncomplementation between sites on different domains (36,48) as well as complementation between sites on the same domain have both also been observed (36). . The IIB domain consists of an antiparallel sheet with strand order ␤1␤2␤4␤3 and three ␣-helices packed against one side of the ␤-sheet. The sequence of the secondary structure elements is ␣1␤1␤2␣2␤3␤4␣3. The active site is at the turn between ␤1 and ␤2 and the solvent-exposed face of ␤2. The figure was produced using RasMol V2.6 (Roger Sayle, Glaxo Wellcome Research and Development).