The Glucose Transporter of Escherichia coli with Circularly Permuted Domains Is Active in Vivo and in Vitro *

The bacterial phosphotransferase system (PTS) consists of two energy-coupling soluble proteins (enzyme I and HPr) and a large number of inner membrane transporters (enzymes II) that mediate concomitant phosphorylation and translocation of sugars and hexitols. The transporters consist of three functional units (IIA, IIB, IIC), which occur either as protein subunits or domains of a multidomain polypeptide. The membrane-spanning IIC domain contains the substrate binding site; IIA and IIB are phosphorylation domains that transfer phosphate from HPr to the transported sugar. The transporter complexes of the PTS are good examples for variation of design by modular assembly of domains and subunits. The domain order is IIC-IIB in the membrane subunit of the Escherichia coli glucose transporter (IICBGlc) and IIB-IIC in Salmonella typhimurium sucrose transporter (IIBCScr). The phosphorylation domain of IICBGlc was translocated from the carboxyl-terminal to the amino-terminal end of the IIC domain, and the activity of the circularly permuted form was optimized by variation of the length and the composition of the interdomain linker. IIBapCGlc with an alanine-proline-rich interdomain linker has 70% of the control specific activity after purification and reconstitution into proteoliposomes. These results indicate that the amino-terminal end of IICBGlc must be on the cytoplasmic side of the inner membrane, that membrane insertion of the IIC domain is insensitive to the modification of its amino-terminal end, and that a domain swap as it could occur by a single DNA translocation event can rapidly lead to a functional protein. However, IIB could not be substituted for by glucokinase. Fusion proteins between the IIC domain and glucokinase do not transport and phosphorylate glucose in an ATP-dependent mechanism, although the IIC moiety displays transport activity upon complementation with soluble subclonal IIB, and the glucokinase moiety retains ATP-dependent nonvectorial kinase activity. This indicates that IIC and IIB are two cooperative units and not only sequentially acting upon a common substrate, and that translocation of glucose must be conformationally coupled to the phosphorylation/dephosphorylation cycle of IIB.

The bacterial phosphotransferase system (PTS) 1 is involved in transport and phosphorylation of carbohydrates and in regulation of the metabolism in response to the availability of these carbohydrates as nutrients (1)(2)(3). The PTS is almost ubiquitous in bacteria but with one exception, has not been found in eukaryotic cells. It consists of two cytoplasmic phosphoryl carrier proteins termed enzyme I and HPr, which sequentially transfer phosphoryl groups from PEP to the different substrate-specific transporters. The transporters comprise three independently folding units of function. IIA and IIB are hydrophilic and contain one phosphorylation site each, and IIC spans the membrane and contains the sugar binding site. The three units occur either as domains in a single polypeptide chain or as subunits of a complex. The number of carbohydratespecific transporters varies from species to species. There are 38 PTS-related genes in Escherichia coli and 26 PTS genes in Bacillus subtilis (EMBL/SwissProt data base), most of which code for PTS transporters and their subunits. Only two open reading frames with homologies to known PTS transporters were found in Hemophilus influenzae (4) and only one in Mycoplasma genitalium (5). The transporters differ in amino acid sequence (from insignificant to more than 90% amino acid similarity), substrate selectivity, and in the structure of the phosphorylation site (histidine and cysteine phosphorylation). Lengeler et al. (6) grouped the transporters into five main families (glucose-sucrose, mannitol-fructose, lactose-cellobiose, mannose, glucitol) according to amino acid sequence similarity.
The EMBL/SwissProt data base lists 24 homologous proteins from 12 different Gram-positive and -negative bacteria in the glucose-sucrose family. 2 Nine have the domain order CB, and 15 have the order BC (Fig. 1). CB and BC can be considered as circularly permuted variants. The hydrophilic B domains contain the strongly conserved sequence N(I/L)X 5 CXTRLRX 4 D with the active-site cysteine, which is phosphorylated by the cognate IIA domains (7). The CB forms, in addition, contain the highly conserved sequence KTPGRED (residues 368 -388 of IICB Glc ). This motif is 35 residues upstream of the active-site cysteine, and it serves as a hinge of constrained mobility between the IIC and the IIB domain (8). It is the only region at which a functional hybrid protein between the homologous transporters for Glc and GlcNAc could be constructed (9). The BC variants have a conserved sequence GNXVXXX(F/Y) or GXGXVXXX(F/Y) 42 residues downstream of the active-site cysteine. It is unique to the BC subgroup and, as judged from the spacing, could be part of a linker between the B and C domains.
The different transporters of the PTS are a good example for variation of design by modular assembly of domains and subunits (10). It has been speculated that the different types of PTS transporters are circularly permuted forms of a consensus structure envisaged to consist of two transmembrane portions and of two large loops, forming cytoplasmic subdomains (11). Saier, Reizer, and co-workers produced a number of sophisticated analyses that suggest that the multidomain proteins of the PTS arose during evolution by repeated recombinational gene rearrangements (12) and document the combinatorial nature and modular design of the PTS transporters (13)(14)(15)(16)(17)(18)(19). Here we show that the C and B domains of of the glucose transporter of E. coli can be circularly permuted with only minor effects on the activity and without detectable effects on membrane insertion.
The wild-type form of the E. coli glucose transporter consists of two subunits IIA Glc and IICB Glc . IIA Glc is a monomeric soluble 18-kDa protein that transfers the phosphate group from HPr to the IICB Glc subunit and at the same time plays an important role in allosteric regulation of adenylate cyclase, glycerol kinase, and carbohydrate transporters for lactose, maltose, and other non-PTS sugars (2). IICB Glc is a homodimeric membrane protein. The 52-kDa subunits consist of two domains. The C domain contains six plus two membranespanning (residues 17-210 and 280 -325), and two extended hydrophilic segments (residues 211-279 and 326 -386) on the cytoplasmic face (20). The B domain on the cytoplasmic face (residues 386 -477) has a split ␣/␤ sandwich fold composed of an antiparallel sheet and three ␣-helices superimposed onto one side of the sheet (21,22). The C domain contains the sugar binding site (9), whereas the B domain contains the phosphorylation site (Cys-421 (7)). This B domain was translocated from its natural location at the carboxyl-terminal end to the amino-terminal end of the C domain. The two domains were coupled by peptide linkers of 4 and 22 residues. The recombinant IIBC Glc subunit with the shorter linker displayed 20%, and the protein with longer Ala-Pro-rich linker (11) displayed 70% of wild-type activity.

EXPERIMENTAL PROCEDURES
Bacterial Strains and Growth Conditions-E. coli K-12 ZSC112L⌬HIC (ptsG manZ glk ptsH ptsI crr) (23) and E. coli K-12 ZSC112L⌬G (ptsG::cat manZ glk) (24) were used as hosts for in vivo complementation assays on McConkey plates and for protein expression. The transformed cells were grown on a rotary shaker in LB broth at 37°C. When the culture had reached A 600 ϭ 1.0, protein expression was induced with 0.1 mM isopropyl-1-thio-␤-D-galactopyranoside, and growth continued overnight.
Plasmid Construction-Plasmids pJFBxC and pJFBapC ( Fig. 2) encode under the control of the Ptac promoter the circularly permuted IIBC Glc variants with a hexahistidine tag at the carboxyl termini. They were constructed from plasmids pJBH and pJCH (24) encoding the IIB and IIC domains, respectively. Plasmid pJBH was digested with Hin-dIII/ScaI, and the insert fragment encoding the B domain was ligated with the vector fragment of pJCH opened with MluI/ScaI (after the ends were made flush with Klenow enzyme). The resulting plasmid contains the DNA for IIB upstream of IIC in the correct orientation but incorrect reading frame. To restore the open reading frame, heteroduplex I (Fig. 2) encoding a linker of four amino acid residues was inserted between BglII and NsiI to afford pJFBxC. pJFBapC was constructed by insertion of the heteroduplex II between AsuII and NdeI in the linker of pJFBxC. Heteroduplex II encodes the 22-residue-long Ala-Pro-rich linker peptide. It was excised from plasmid pTSL23 (25). Plasmids In the sucrose family, the domain order is IIB (diagonal hatching) IIC (vertical hatching). In IICB Glc (ptsG) and related transporters of the glucose family, the domain order is IIC followed by IIB. The linker consensus sequences are indicated in singleletter amino acid code. The domain order of the circularly permuted form of the glucose transporter (IIBapC and IIBxC) and the fusion proteins between the IIC domain and glucokinase (cross-hatching) are shown together with the sequences of the short (x) and the long alanineproline-rich (ap) linkers. Lowercase letters refer to the last three residues of IIC and the first three residues of glucokinase. pJFCK, pMSKxC, and pMSKapC encode fusion proteins between the IIC domain and glucokinase (K) with hexahistidine tags at the carboxyl termini. They were constructed from plasmids pJCH, pJFBxC, pJF-BapC, and pMSGlk. pMSGlk was constructed as follows. The coding sequence for glucokinase was polymerase chain reaction-amplified with primers AGCATACATATGACAAAGTATGCATTACTC and TACGAT-GCATGCTTATTCGAACAGAATGTGACCTAAGGTCTG (restriction sites are underlined) using plasmid pCSF2 (gift of W. Boos (26)) as template. The polymerase chain reaction product was digested with NdeI and SphI purified and cloned into pMS470⌬8, an expression vector derived from pJF118EH (27) containing Ptac and a ribosome binding sequence upstream of an NdeI site. pJFCK was constructed as follows. The coding sequence for glucokinase was polymerase chain reaction-amplified with primers AGCATACCCGGGTGACAAAGTATG-CATTACTC and TACGATAGATCTCAGAATGTGACCTAAGGTCTG, digested with SmaI and BglII, purified, and cloned into pJCH (28) opened at SmaI and BglII. pMSKxC and pMSKapC were constructed as follows. pJFBxC and pJFBapC were digested with AsuII and ScaI, and the insert fragments (coding for the xC and apC domain, respectively, and the amino-terminal half of ␤-lactamase) were ligated with the vector fragment of pMSGlk (encoding glucokinase and the carboxylterminal half of ␤-lactamase) obtained by digestion with AsuII and ScaI. Plasmid pACYCB encodes the IIB Glc domain under the control of the Ptac promoter, lacI q , and kanamycin resistance. It was constructed as follows. Plasmid pJBH was digested with NdeI and ScaI and blunted with Klenow enzyme. Plasmid pACYC177 (29) was digested with BamHI and ScaI and blunted with Klenow enzyme. The vector fragment of pACYC177 and the insert fragment of pJBH were ligated. The recombinant plasmid was digested with PvuI (partial) and ScaI, and the fragment with the deletion within bla was self-ligated to afford pACYCB. Plasmid pJFGC421S was constructed from plasmid pTSG4 -421 (30) and pTSGH11 (8).
Assay for Phosphoenolpyruvate:Sugar Phosphotransferase Activity-Sugar phosphorylation activity was assayed by the ion exchange method (31,32). A cell-free cytoplasmic extract from E. coli ZSC112L⌬G(pTSHIC9) overexpressing enzymes I, HPr, and IIA Glc or the proteins purified from this extract were used to complement phosphotransferase activity (33). Either Glc or ␣-methyl-D-glucopyranoside (specific activities 1000 dpm/nmol) were used as substrates.
Assay for Vectorial Import into Proteoliposomes-Purified IICB Glc was reconstituted with E. coli lipids, and glucose uptake was assayed as described (34).
Other Techniques-Protein samples were not boiled in sample buffer before electrophoresis on standard 15% polyacrylamide gels (32). Gels were stained with Coomassie Blue. Protein concentrations were determined by a modified Lowry assay (35) with bovine serum albumin as the standard.

RESULTS
Gene Reconstruction and Protein Purification-The genes for two circularly permuted IIBC Glc variants of the glucose transporter (IICB Glc ) were reconstructed from the ptsgG gene fragments encoding the two domains and two heteroduplexes encoding linker sequences of different length and amino acid composition ( Fig. 2A). Both IIBxC Glc and IIBapC Glc could be solubilized quantitatively with 2% N-decyl-␤-D-maltopyranoside. The membrane extracts were adsorbed to Ni 2ϩ -NTA resin, and IIBC Glc was eluted with imidazole. We observed that phosphotransferase activity was best preserved when the samples were dialyzed against phosphate buffer immediately after elution. The imidazole-free proteins could be kept in pentaeth- ylene glycoloctyl ether for 3 weeks at 4°C until they started to loose activity. Freezing at Ϫ20°C resulted in precipitation and partial inactivation. About 55% of the phosphotransferase activity originally present in the membranes was recovered after metal affinity chromatography. Part of the activity was lost due to incomplete binding to the column. Between 1.5 and 2 mg of pure IIBC Glc could be recovered from 1 liter of an overnight cell culture. IIBxC Glc (M r 53,229) with the short linker had an increased electrophoretic mobility (Fig. 2B) relative to wildtype IICB Glc (M r 51,864), suggesting that it binds more SDS and/or has a different shape in the partially unfolded form. Attempts to completely unfold the proteins by boiling in sample buffer resulted in protein aggregation. Proteolytic degradation can be excluded as the cause of the increased mobility since Edman degradation demonstrated an intact amino terminus (results not shown), and the intactness of the carboxyl terminus can be inferred from successful binding to the Ni 2ϩ -NTA column. IIBapC Glc (M r 54,768) with the 22 residues linker rich in alanine and proline has an only marginally decreased electrophoretic mobility.
Phosphotransferase Activity of the Mutants with Circularly Permuted Domains-E. coli ZSC112L⌬G expressing IIBapC Glc and wild-type IICB Glc under the control of the leaky Ptac promoter ferment glucose on McConkey plates already without induction (results not shown). In contrast, cells expressing IIBxC Glc ferment glucose only after induction of gene expression with 10 M isopropyl-1-thio-b-D-galactopyranoside, indicating that IIBxC Glc with the short linker is less active than its counterpart with the longer linker and wild-type IICB Glc . This difference was confirmed by in vivo transport assays. E. coli expressing IIBxC Glc displayed a reduced transport activity at all levels of induction (Fig. 3A).
IICB Glc catalyzes vectorial transport coupled to phosphorylation of the substrate (vectorial phosphorylation) as well as substrate phosphorylation without transport (nonvectorial phosphorylation). Active transport was measured with IICB Glc containing proteoliposomes that were loaded with PEP, enzyme I, HPr, and IIA Glc and to which glucose was added from the outside. The initial rates of glucose transport into these proteoliposomes were similar for wild-type IICB Glc , IIBxC Glc , and IIBapC Glc , but the level to which glucose was accumulated was only 55% for IIBxC Glc and 75% for IIBapC Glc (Fig. 3B). Nonvectorial phosphorylation was measured with IICB Glc -containing proteoliposomes to which glucose, PEP, and the soluble components were all added from the outside. Purified IIBxC Glc displayed between 20 and 30% wild-type nonvectorial PTS activity in the presence of saturating concentrations of enzyme I, HPr, IIA Glc , and glucose. IIBapC Glc had between 50 and 70% wild-type activity (Fig. 3C).
It is not clear whether docking of IIA Glc to the IIB domain, phosphate transfer between IIA and IIB and between IIB and Glc, or translocation of Glc is affected by the circular permutation. Comparing the K m for IIA Glc of the two forms allows estimation of differences of affinity for IIA Glc . IIA Glc was titrated in the presence of excess EI and HPr, and a rate-limiting concentration of IICB Glc and IIBxC Glc , respectively. The K m for IIA Glc determined as IIA Glc concentration necessary for half maximal phosphotransferase activity was 2.3 Ϯ 0.5 M for wild-type IICB Glc and 0.36 Ϯ 0.8 M for IIBxC Glc (Fig. 4). Assuming that the overall rate of phosphate transfer from IIA to Glc is faster than the dissociation rate of the IIA⅐IIB complex, the reduced K m can be accounted for by the observed reduction of V max (Fig. 3C). Therefore it appears that the affinity of IIA for IIBxC Glc is comparable with the affinity to wild-type IICB Glc .
Substitution of the IIB Domain by Glucokinase-The comparable activities of IICB Glc and IIBapC Glc could suggest that the main function of IIB is to remove by phosphorylation the tightly bound glucose from the cytoplasmic face of the IIC domain and that covalent binding of IIB to IIC would serve to maintain a high concentration of the phosphoryl donor at the glucose binding site. If this were so, glucokinase (Glk) might be capable of complementing IIB activity. To test this hypothesis, fusions of the 35-kDa E. coli glucokinase (26) to the amino and carboxyl terminus of the IIC domain were constructed, and the fusion proteins were expressed in E. coli ZSC112⌬HIC lacking glucokinase, IICB Glc , and IIA Glc as well as the general PTS proteins enzyme I and HPr. The transformants formed yellow colonies on McConkey glucose indicator plates containing different isopropyl-1-thio-b-D-galactopyranoside concentrations. To test for function of the IIC domain, the fusion proteins were expressed in ZSC112⌬G(pACYCB). This strain lacks IICB Glc and glucokinase but expresses the chromosomally encoded enzyme I, HPr, IIA Glc , and the plasmid-encoded subclonal IIB Glc domain. Cells coexpressing IIC-Glk and subclonal IIB Glc formed red colonies on McConkey glucose plates, whereas cells expressing GlkxIIC or GlkapIIC and IIC Glc were red-centered, indicating that the IIC domain of the fusion protein was functional.
Since the fermentation phenotype on McConkey plates was TABLE I Expression and subcellular distribution of glucokinase and glucokinase-II Glc fusion proteins Cells from 800-ml overnight cultures were fractionated, and glucokinase activity was determined. 100% activity corresponds to 1.29 ϫ 10 9 nmol of Glc 6-phosphate formed in 30 min at 37°C. The total activity of the fusion proteins in percent of the activity of wild-type glucokinase is given in the last column. After centrifugation, the supernatant was withdrawn by aspiration. The top 90% of the supernatant and the 10% of the bottom layer over the membrane sediment were collected separately. variable and did not allow for quantitative analysis of function, lysates from cells expressing Glk, IIC-Glk, GlkxIIC, and Glka-pIIC were fractionated, and the membrane and cytoplasmic fractions were assayed for glucokinase and IIC-dependent phosphotransferase activity (Tables I and II). The IIC-dependent phosphotransferase activity was assayed with membranes in the presence of increasing concentrations of purified IIB Glc (Fig. 5, A and B). The C421S mutant of IICB Glc was used as a reference for a protein with an inactive B domain but otherwise unchanged structure. The results are summarized in Table II.
The IIC activity predominantly is in the membrane fraction. IIC-Glk has a 10 times higher specific phosphotransferase activity than GlkapIIC, whereas GlkxIIC is inactive. The differences of specific phosphotransferase activity between IIC-Glk and GlkapIIC in crude membranes reflect the different expression levels of the two fusion proteins and not different specific activities, as suggested by the yields of purified proteins (Fig.  2B, lanes 4 and 5). The Glk activity of wild-type Glk was localized in the cytoplasmic fraction, whereas the Glk activity of the fusion proteins GlkapIIC and IIC-Glk was in the membrane fraction (Table I). Unexpectedly, glucokinase activity from cells expressing GlkxIIC was found in the cytoplasmic fraction. It is not clear whether this activity in the soluble fraction originates from full-length protein that did not insert into the membrane or from a soluble proteolytic breakdown product. The specific glucokinase activities of the purified IIC-Glk and GlkapIIC fusion proteins differ by a factor of two (Table III). Cells expressing glucokinase fusion proteins produced between 0.1 and 1.0% wild-type glucokinase activity (Table I). This 1% activity ratio of membrane bound to wildtype cytoplasmic glucokinase activity is of the same order as the 3% volume ratio of inner membrane to cytoplasmic compartment.

DISCUSSION
The PTS transporters of the glucose family consist of two domains, the transmembrane IIC domain and the cytoplasmic IIB domain. The domain order can be either CB as in the glucose transporter or BC as in the sucrose transporter IIBC Scr of Salmonella typhimurium (36). Here we show that the two domains of the IICB Glc subunit of the glucose transporter can be circularly permuted, thus mimicking domain shuffling as it might have occurred during the evolution of the PTS transporters from common ancestors. The circularly permuted form (IIB-C Glc ) complemented glucose transport and phosphorylation in vivo and could be purified and functionally characterized in vitro. The permuted form without additional structural adjustments had already between 10 and 20% wild-type activity. Increasing the length of the interdomain linker from 4 to 20 residues (Fig. 1) increased the activity to 70% of the wild-type reference. At the same time, the phosphorylation domain IIB could not be functionally substituted for by glucokinase. This indicates that IIC and IIB are two cooperative units and not only two proteins that sequentially act upon a common substrate and that translocation of glucose must be conformationally coupled to the phosphorylation/dephosphorylation cycle of IIB. However, despite the high specificity of the interaction between IIC and IIB, binding is not so tight that the covalently bound but inactive IIB cannot be displaced. For example, the soluble subclonal IIB Glc unit (10 kDa) can successfully displace an inactive IIB domain (in the C421S mutant of IICB Glc , Table  II) or the even larger glucokinase moiety (35 kDa) in a fusion protein between the IIC domain and glucokinase. Coupling between the two domains is likely to be mediated by parts of the antiparallel ␤-sheet, which carries the active site Cys-421 and the essential Arg-424 and Arg-426 (8). The helices that form a dome-like structure on the face of the antiparallel ␤-sheet opposite to the active site (21) mediate the covalent links to the neighboring domains in multidomain proteins. In IICB Glc , helix 1 connects the ␤-sheet to the carboxyl terminus of the IIC domain, whereas helix 3 is free. In the homologous transporter for GlcNAc (IICBA GlcNAc ) (34), helix 3 connects the ␤-sheet with the IIA domain. This same helix 3 is able to link the sheet to the IIC domain in the circularly permuted form of the IICB Glc subunit.
The successful fusion of a cytoplasmic domain to the amino terminus of the IIC Glc domain confirms the prediction from PhoA and LacZ fusion experiments that the amino terminus of IICB Glc is located on the cytoplasmic face of the membrane (20).   16 18 However, insertion of the IIC fusion protein with modified amino-terminal ends is less efficient than the insertion of fusion proteins with the native amino terminus. Insertion is also less efficient with increasing size of the attached domain, but this inhibitory effect partially can be compensated by increasing the length of the interdomain linker. The interdomain linker was chosen from among the well characterized alanine-proline-rich sequences that form flexible hinges in multidomain proteins (37)(38)(39). They occur naturally in the E2 chain of the pyruvate dehydrogenase, in the proteolytic processing site of the endo-␤-N-acetylglucosaminidase preprotein, and in the IIAB Man subunit of the E. coli mannose transporter (summarized in Ref. 40). This type of linker has been successfully used to construct a multidomain protein from the four subunits of the glucose PTS (23). The length of the linker appears important for membrane insertion of the IIC domain.
When transformants expressing soluble glucokinase or IICglucokinase fusion proteins were plated on McConkey plates containing maltose (a non-PTS sugar), only cells expressing IIC-Glk formed strongly fermenting colonies, whereas cells expressing soluble glucokinase and GlkapIIC remained yellow (results not shown). Meyer et al. (26) report that high concentrations of soluble glucokinase interfered with the expression of the maltose system. It is likely that expression of soluble glucokinase from a multicopy plasmid resulted in a nonfermenting phenotype, whereas the approximately 100-fold reduced glucokinase activity of IIC-Glk was just right for the phosphorylation of intracellular glucose but did not yet repress the maltose system. The 3 to 10 times lower glucokinase activity of GlkxIIC and GlkapIIC might be insufficient for maltose fermentation under the chosen conditions.
The results of this study indicate that a domain swap as it could occur by a single DNA translocation event can immediately lead to a functional protein and that only minor structural adjustments, such as duplicating the fusion joint, might be necessary to compensate for a partial loss of activity due to a structural mismatch between the reoriented domains.