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• W2036830304 abstract "The Escherichia coli BglF protein, a permease of the phosphoenolpyruvate-dependent phosphotransferase system, catalyzes transport and phosphorylation of β-glucosides. In addition, BglF regulates bgl operon expression by controlling the activity of the transcriptional regulator BglG via reversible phosphorylation. BglF is composed of three domains; one is hydrophobic, which presumably forms the sugar translocation channel. We studied the topology of this domain by Cys-replacement mutagenesis and chemical modification by thiol reagents. Most Cys substitutions were well tolerated, as demonstrated by the ability of the mutant proteins to catalyze BglF activities. Our results suggest that the membrane domain contains eight transmembrane helices and an alleged cytoplasmic loop that contains two additional helices. The latter region forms a dynamic structure, as evidenced by the alternation of residues near its ends between faced-in and faced-out states. We suggest that this region, together with the two transmembrane helices encompassing it, forms the sugar translocation channel. BglF periplasmic loops are close to the membrane, the first being a reentrant loop. This is the first systematic topological study carried out with an intact phosphotransferase system permease and the first demonstration of a reentrant loop in this group of proteins. The Escherichia coli BglF protein, a permease of the phosphoenolpyruvate-dependent phosphotransferase system, catalyzes transport and phosphorylation of β-glucosides. In addition, BglF regulates bgl operon expression by controlling the activity of the transcriptional regulator BglG via reversible phosphorylation. BglF is composed of three domains; one is hydrophobic, which presumably forms the sugar translocation channel. We studied the topology of this domain by Cys-replacement mutagenesis and chemical modification by thiol reagents. Most Cys substitutions were well tolerated, as demonstrated by the ability of the mutant proteins to catalyze BglF activities. Our results suggest that the membrane domain contains eight transmembrane helices and an alleged cytoplasmic loop that contains two additional helices. The latter region forms a dynamic structure, as evidenced by the alternation of residues near its ends between faced-in and faced-out states. We suggest that this region, together with the two transmembrane helices encompassing it, forms the sugar translocation channel. BglF periplasmic loops are close to the membrane, the first being a reentrant loop. This is the first systematic topological study carried out with an intact phosphotransferase system permease and the first demonstration of a reentrant loop in this group of proteins. The Escherichia coli BglF protein (EIIbgl), an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS), 1The abbreviations used are: PTS, phosphoenolpyruvate-dependent phosphotransferase system; TM, transmembrane helix; ISO membranes, membrane vesicles with inside-out orientation; pNPG, p-nitrophenyl β-d-glucopyranoside; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide; MTSET, [2-(trimethylammonium) ethyl]methanethiosulfonate bromide; MTSES, sodium(2-sulfonatoethyl)methanethiosulfonate; MTS, methanethiosulfonate; TCEP, tris(2-carboxyethyl)phosphine; DM, n-dodecyl β-d-maltoside; 5-FM, fluorescein 5-maleimide; P-, phospho. catalyzes concomitant transport and phosphorylation of β-glucosides across the cytoplasmic membrane (1Fox C.F. Wilson G. Proc. Natl. Acad. Sci. U. S. A. 1968; 59: 988-995Crossref PubMed Scopus (61) Google Scholar). In addition to its ability to phosphorylate its sugar substrate, BglF phosphorylates the transcriptional regulator BglG in the absence of β-glucosides and dephosphorylates P-BglG upon addition of β-glucosides to the growth medium (2Amster-Choder O. Houman F. Wright A. Cell. 1989; 58: 847-855Abstract Full Text PDF PubMed Scopus (93) Google Scholar, 3Amster-Choder O. Wright A. Science. 1990; 249: 540-542Crossref PubMed Scopus (55) Google Scholar). By controlling the phosphorylation state of BglG, BglF controls the dimeric state of BglG and, thus, its ability to bind RNA and antiterminate transcription of the bgl operon (4Amster-Choder O. Wright A. Science. 1992; 257: 1395-1398Crossref PubMed Scopus (110) Google Scholar). BglF dimerizes spontaneously in the membrane, and its dimeric form can catalyze all the above mentioned activities (5Chen Q. Amster-Choder O. Biochemistry. 1998; 37: 8714-8723Crossref PubMed Scopus (16) Google Scholar). Several other enzymes II of PTS were shown to regulate activity of transcription factors. Two representative examples are SacX, a sucrose permease from Bacillus subtilis, which regulates the activity of the BglG homologue SacY by reversible phosphorylation (6Crutz A.M. Steinmetz M. Aymerich S. Roselyne R. Le Cog D. J. Bacteriol. 1990; 172: 1043-1050Crossref PubMed Google Scholar, 7Idelson M. Amster-Choder O. J. Bacteriol. 1998; 180: 660-666Crossref PubMed Google Scholar), and PstG, a glucose permease from E. coli, which regulates the activity of the global regulator Mlc by membrane sequestration (8Lee S. Boos W. Bouche J. Plumbridge J. EMBO J. 2000; 19: 5353-5361Crossref PubMed Scopus (122) Google Scholar, 9Tanaka Y. Kimata K. Aiba H. EMBO J. 2000; 19: 5344-5352Crossref PubMed Scopus (124) Google Scholar). The phosphate flux in PTS starts with a phosphoryl group, donated by phosphoenolpyruvate, which is passed through the general PTS proteins, enzyme I, and HPr, to the various sugar-specific permeases. Like many other PTS permeases, BglF is composed of three domains: the hydrophilic A and B domains (IIAbgl and IIBbgl) and the hydrophobic C domain (IICbgl) (reviewed in Refs. 10Postma P.W. Lengeler J.W. Jacobson G.R. Microbiol. Rev. 1993; 57: 543-594Crossref PubMed Google Scholar, 11Lengeler J.W. Jahreis K. Wehmeier U.F. Biochim. Biophys. Acta. 1994; 1188 (and references therein): 1-28Crossref PubMed Scopus (128) Google Scholar, 12Robillard G.T. Broos J. Biochim. Biophys. Acta. 1999; 1422: 73-104Crossref PubMed Scopus (98) Google Scholar). The domains of BglF are covalently linked to one another in the order BCA. The A domain is phosphorylated by HPr; the phosphate is then transferred to the B domain and subsequently to β-glucosides; the membrane-spanning C domain presumably forms the sugar translocation channel and at least part of the sugar-binding site. Cys-24, residing in the B domain, was shown to be responsible for the phosphorylation of both the incoming sugar and the BglG protein and for the dephosphorylation of P-BglG (13Chen Q. Arents J.C. Bader R. Postma P. Amster-Choder O. EMBO J. 1997; 16: 4617-4627Crossref PubMed Scopus (55) Google Scholar, 14Chen Q. Postma P.W. Amster-Choder O. J. Bacteriol. 2000; 182: 2033-2036Crossref PubMed Scopus (12) Google Scholar). The mechanism that ensures correct delivery of the phosphoryl group to the right entity, sugar or protein, by the same active site is not known. A clue to this mechanism seems to lie in the different domain requirements of the distinct functions; whereas the B domain is sufficient for BglG phosphorylation, both the B and C domains, connected in the order BC, are required for the sugar-stimulated functions, i.e. sugar phosphotransfer and P-BglG dephosphorylation (15Chen Q. Amster-Choder O. Biochemistry. 1998; 37: 17040-17047Crossref PubMed Scopus (11) Google Scholar, 16Chen Q. Amster-Choder O. J. Bacteriol. 1999; 181: 462-468Crossref PubMed Google Scholar). Catalysis of the sugar-stimulated functions involves specific interaction(s) between the active site-containing B domain and the integral membrane C domain (17Chen Q. Nussbaum-Shochat A. Amster-Choder O. J. Biol. Chem. 2001; 276: 44751-44756Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). To understand better how BglF catalyzes its different activities, we investigated its membrane topology. To examine the way BglF traverses through the membrane, we constructed a set of single-Cys BglF variants by site-directed mutagenesis, and we assessed the exposure and the sidedness of the planted cysteines, in whole cells and in membrane vesicles with an inside-out orientation, by chemical modification with thiol-specific reagents. Of the 36 constructed single-Cys mutants, only one was not expressed. Because the BglF-active site is a cysteine residue (Cys-24Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9433) Google Scholar), single-Cys mutants are not active. Hence, we constructed a parallel set of di-Cys mutants, each containing Cys-24 in the B domain and a single cysteine in the membrane C domain. We used the di-Cys mutants to test the effect of the Cys replacements on BglF activities. All mutants catalyzed phosphorylation and dephosphorylation of BglG, and except for one, they were all capable of β-glucosides phosphotransfer, implying that they retained proper folding. The topology of single- and di-Cys BglF variants was studied by the use of membrane-permeant and impermeant thiol-binding reagents. Most significantly, the experiments performed with the two sets of mutants led to the same conclusions, indicating that the mutation in the B domain does not affect the folding and the membrane topology of the BglF variant proteins. Based on the results presented here, we suggest that BglF contains eight transmembrane helices (TMs I-VIII). The periplasmic loops are close to the membrane, the first being a reentrant loop. The region between TM VI and TM VII, an alleged cytoplasmic loop, is rather sizeable. It contains a pair of putative α-helices and positions near its termini flip occasionally between faced-in and faced-out states. The accessibility to one position in this region is blocked by the sugar substrate. We suggest that this region and the two TMs encompassing it form the sugar translocation channel. Our study is the first detailed topological study of a PTS sugar permease in the context of the intact protein. Previous systematic topological studies of PTS permeases were performed with fusion proteins between progressively or randomly truncated PTS permeases and β-galactosidase or alkaline phosphatase (reviewed in Ref. 18Siebold C. Flukiger K. Beutler R. Erni B. FEBS Lett. 2001; 504: 104-111Crossref PubMed Scopus (93) Google Scholar). These studies also suggested the existence of a large cytoplasmic loop, which is likely to be involved in sugar phosphotransfer. The actual dynamics of this region between the cytoplasm and periplasm is manifested here for the first time. Chemicals—Isopropyl 1-thio-β-d-galactopyranoside, n-dodecyl β-d-maltoside (DM), p-nitrophenyl β-d-glucopyranoside (pNPG), salicin, arbutin, imidazole, lysozyme, DNase, and RNase were obtained from Sigma. 4-(2-Aminoethyl)benzenesulfonyl fluoride was obtained from Calbiochem. Nickel-nitrilotriacetic acid resin was obtained from Qiagen. Fluorescein 5-maleimide (5-FM) was obtained from Pierce. 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), [2-(trimethylammonium) ethyl]methanethiosulfonate bromide (MTSET), and sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) were obtained from Toronto Research Chemicals Inc. Anhydrotetracycline hydrochloride was obtained from Acros Organics. Strains—The following E. coli K12 strains were used: AG1688 (MC1061 (F′128lacIq lac::Tn5)) was used for the construction of single- and di-Cys BglF mutants. HMS174(DE3) (F-recA1 hsdR(rK12-mK12+) RifR (DE3)), obtained from Novagen, was used for monitoring expression of the BglF variants as His-tagged proteins from pET15b and for assaying sugar phosphotransfer and accessibility of the planted cysteines to thiol reagents. MA200-1, which carries a bgl′-lacZ fusion on its chromosome (λ bglR7 bglG′ lacZ+lacY+) and a defective bglF gene (19Mahadevan S. Reynolds A.E. Wright A. J. Bacteriol. 1987; 169: 2570-2578Crossref PubMed Google Scholar), was used for assaying BglG regulation by the BglF variants. Plasmids—Plasmid pT7OAC-F, which carries the entire bglF gene cloned downstream of the phage T7 late promoter in pT712 (2Amster-Choder O. Houman F. Wright A. Cell. 1989; 58: 847-855Abstract Full Text PDF PubMed Scopus (93) Google Scholar), was used as a reagent to construct two plasmids encoding BglF variants, one devoid of Cys codons (Cys-less) and the other containing the active site cysteine as a sole cysteine (Cys-24 only). In both variants the native cysteines were replaced by serines. These two plasmids were used as reagents to construct two parallel sets of plasmids encoding BglF mutants with either single- or di-cysteines, respectively, each set comprising 36 mutants with Cys substitutions as specified under “Results.” All mutations were introduced by site-directed mutagenesis, which was carried out by overlap extension with PCR (20Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6869) Google Scholar). The mutations introduced or eliminated sites for restriction enzymes, which were useful during the screening for the mutant plasmids. All mutations were confirmed by sequencing. The bglF alleles encoding the single- and di-Cys variants were cloned in pET15b, to generate N-terminal His-tagged BglF variants. More details on plasmid constructions are available from the authors upon request. pZGM-B, obtained from G. Monderer-Rothkoff, was used for the expression of the bglB gene required for assaying salicin phosphotransfer. Induction of the bglB gene was achieved by the addition of 100 ng of anhydrotetracycline hydrochloride to the growth medium. β-Glucoside Phosphotransfer—To estimate qualitatively the ability of the BglF variants to transfer β-glucosides into the cells while phosphorylating them, HMS174(DE3) cells expressing the di-Cys BglF mutants were plated on MacConkey-arbutin and MacConkeysalicin plates. Utilization of the β-glucosides arbutin and salicin depended on the ability of the plasmid-encoded BglF variants to transport and phosphorylate these sugars, which were then cleaved by the products of the constitutively expressed chromosomal bglA gene and the plasmid-encoded bglB gene, respectively. Hence, the formation of red colonies on MacConkey-arbutin and MacConkeysalicin plates was indicative of the ability of the di-Cys BglF mutants to catalyze β-glucosides phosphotransfer. To estimate quantitatively the ability of the BglF variants to phosphotransfer β-glucosides, the assay described by Schaefler (21Schaefler S. J. Bacteriol. 1967; 93: 254-263Crossref PubMed Google Scholar) was carried out with minor modifications. HMS174(DE3) cells expressing the di-Cys BglF mutants were grown in M63 minimal medium, supplemented with 0.4% succinate as a carbon source, at 37 °C. When cells reached A600 = 0.4-0.5, isopropyl 1-thio-β-d-galactopyranoside was added to a final concentration of 1 mm. After 30 min, cultures were put on ice for 20 min, and their absorbance was measured (A600). Cells (1 ml) were pelleted by centrifugation, washed, and resuspended in 0.9 ml of 0.075 m phosphate buffer, pH 7.5, containing 1 mm MgSO4. The reaction was started by adding 0.1 ml of the β-glucoside pNPG at a final concentration of 20 mm. After 10 min at 37 °C, the reaction was stopped by the addition of 0.5 ml of 2 m Na2CO3. Cells were pelleted by centrifugation, and the amount of p-nitrophenol was estimated by measuring absorbance at 410 nm. The units of enzyme activity were calculated using the following equation: units = (A410 × 1000)/(A600 × 1 ml × 10 min). Cysteine Accessibility to Thiol Reagents in Whole Cells—Cysteine accessibility to thiol reagents was assayed essentially as described by Ninio et al. (22Ninio S. Elbaz Y. Schuldiner S. FEBS Lett. 2004; 562: 193-196Crossref PubMed Scopus (48) Google Scholar) with some modifications. HMS174(DE3) cells expressing single- and di-Cys BglF variants were grown in LB medium supplemented with 0.1 mg/ml ampicillin at 30 °C. When cells reached A600 = 0.3, expression of the BglF variants was induced by the addition of isopropyl 1-thio-β-d-galactopyranoside to a final concentration of 0.1 mm. After 1.5 h, 4 aliquots of 1 ml of culture were pelleted by centrifugation, washed with Tris-NaCl buffer (150 mm NaCl, 15 mm Tris, pH 7.5), pelleted again, and resuspended in 200 μl of Tris-NaCl buffer. Freshly made MTSET, MTSES, or MTSEA, at final concentrations of 1, 1, and 2.5 mm, respectively, were added, each to 1 aliquot. The fourth aliquot served as a control and hence was not treated with MTS reagents. After 10 min of incubation at either 25 or 4 °C, as indicated in the text, cells were pelleted, washed with Tris-NaCl buffer, and pelleted again. The cells were lysed by resuspending them in 100 μl of 30 mm Tris-HCl, pH 8.0, 30% sucrose, 10 mm EDTA, pH 8.0, and 50 μg/ml lysozyme and incubating them for 15 min at 37 °C. After the addition of 600 μl of 15 mm MgSO4 containing 5 μg/ml DNase, incubation was continued for 15 min at 37 °C. Membranes were pelleted by centrifugation (14,000 rpm for 15 min at 4 °C) and resuspended in 100 μl of Tris-NaCl buffer. Membranes were solubilized by the addition of DM to a final concentration of 0.8% and gentle rotation of the tubes for 15 min at room temperature. The BglF variant proteins were purified to near-homogeneity by using nickel-chelate chromatography as follows. To each aliquot, 300 μl of Tris-NaCl buffer containing 30 mm imidazole and 50 μl of nickel-nitrilotriacetic acid beads (formerly washed twice with Tris-NaCl buffer and once with the Tris-NaCl buffer containing 0.08% DM and 30 mm imidazole) were added, and the tubes were rotated for 1 h. Subsequently, the beads were washed with denaturation buffer (Tris-NaCl buffer containing 6 m urea and 0.5% SDS), pelleted, and resuspended in 100 μl of denaturation buffer containing 0.25 mm fluorescein 5-maleimide (5-FM, dissolved in Me2SO). Tubes were rotated for 20 min at room temperature, and excess of 5-FM was quenched by the addition of 300 μm denaturation buffer containing 5 mm β-mercaptoethanol for 3 min. The beads were pelleted, and the His-tagged BglF proteins were eluted by incubating the beads for 10 min at 30 °C in 28 μl of Laemmli buffer containing 0.07 m EDTA, pH 8.0. Following a short spin, samples were analyzed on 10% SDS-polyacrylamide gels. Fluorescent profiles were recorded with ImageMaster VDS-CL imaging system (UV 312 nm, filter band pass 520 nm). The protein content in each lane was evaluated by staining the same gel with Coomassie Brilliant Blue. The Coomassie-stained profile was recorded with ImageMaster VDS-CL imaging system (white light, no filter). The amounts of the fluorescent BglF bands were normalized to the amounts of the stained bands using the TINA 2.0 software. The percentage of labeling of each mutant protein with 5-FM after treatment with MTS reagents was assessed relative to the 5-FM labeling of the same protein that has not been treated with MTS reagents, which was defined as 100%. The percentage of inhibition of 5-FM labeling by the MTS reagents was calculated as 100% minus the percentage of 5-FM labeling. The results obtained with the untreated samples ruled out the possibility that free cysteines are oxidized in this procedure after cell lysis. Preparation of Membrane Vesicles with an Inside-out Orientation and Cys Accessibility Analyses—HMS174(DE3) cells expressing single-Cys BglF variants were grown and induced as described above, and inside-out (ISO) membrane vesicles were prepared essentially as described before (23Rosen B.P. Methods Enzymol. 1986; 125: 328-336Crossref PubMed Scopus (114) Google Scholar) with some modifications. Cells were washed with 50 mm Tris-HCl, pH 7.5, 5 mm MgCl2, and 0.2 mm 4-(2-aminoethyl)benzenesulfonyl fluoride and resuspended in 10 mm Tris-HCl, pH 7.5, 140 mm choline chloride, 0.25 m sucrose, 5 mm MgSO4, and 5 μg/μl DNase. The suspension was passed once through a French press cell operated at 10,000 pounds/square inch. Unbroken cells were removed by centrifugation at 5,000 × g for 15 min at 4 °C, and membranes were collected from the supernatant by centrifugation at 100,000 × g for 1 h at 4 °C. The membranes were frozen in liquid nitrogen and stored at -80 °C in 50 mm potassium phosphate, pH 7.4, 5 mm MgSO4 and 10% glycerol at a protein concentration of 15-20 mg/ml. ISO membranes were incubated for 2 min at 25 °C in Tris-NaCl buffer (see above) in the presence or absence of MTSET at a final concentration of 0.01 mm and washed twice with Tris-NaCl buffer. The BglF protein variants were purified and labeled with 5-FM as described above for whole cells. Membrane Topology Prediction Methods—Five topology prediction methods, TMHMM (24Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9433) Google Scholar), HMMTOP (25Tusnady G.E. Simon I. J. Mol. Biol. 1998; 283: 489-506Crossref PubMed Scopus (952) Google Scholar), MEMSAT (26Jones D.T. Taylor W.R. Thornton J.M. Biochemistry. 1994; 33: 3038-3049Crossref PubMed Scopus (714) Google Scholar), TOPPRED (27von Heijne G. J. Mol. Biol. 1992; 225: 487-494Crossref PubMed Scopus (1408) Google Scholar), and PHD (28Rost B. Fariselli P. Casadio R. Protein Sci. 1996; 5: 1704-1718Crossref PubMed Scopus (535) Google Scholar), were used in their single-sequence mode. All user-adjustable parameters were left at their default values. All these methods are available on-line. Prediction of Transmembrane Segments and Construction of Single- and Di-cysteine BglF Mutants—We used five topology prediction algorithms, HMMTOP, TMHMM, TOPRED, MEMSAT, and PHD (see “Experimental Procedures”), to predict the membrane topology of BglF (Fig. 1, first five lines). The extent of agreement between these frequently used prediction methods, provided by a simple “majority vote” approach, was shown to be a good indication for the reliability of the predicted topology of E. coli membrane proteins (29Nilsson J. Persson B. von Heijne G. FEBS Lett. 2000; 486: 267-269Crossref PubMed Scopus (94) Google Scholar). The consensus prediction approach was extended to also include cases where only part of the global topology was covered by the agreement (30Nilsson J. Persson B. Von Heijne G. Protein Sci. 2002; 11: 2974-2980Crossref PubMed Scopus (38) Google Scholar). The five algorithms predicted that the C domain of BglF contains 7-12 transmembrane (TM) helices. As shown in Fig. 1 and summarized in Table I, the different methods give very similar predictions for the location of the first five TMs (five out of five for helices I, II, and IV and four out of five for helices III and V) and for the last TM (five out of five). The five methods also predicted the existence of two helices in the middle, with a high degree of agreement on their borders (designated i and ii in Fig. 1). It was argued that the fraction of correctly predicted topologies of E. coli inner membrane proteins is close to one when four or more of these methods agree (29Nilsson J. Persson B. von Heijne G. FEBS Lett. 2000; 486: 267-269Crossref PubMed Scopus (94) Google Scholar). We made use of these predictions to choose residues, which were predicted with a high degree of certainty to face either the cytoplasm or the periplasm, and others, on which there is no consensus between the predictions, and we mutated them to cysteines in a background of a Cys-less BglF derivative. In addition, we constructed variants that contain each of the native cysteines in BglF as a single cysteine, including one in the B domain and one in the linker connecting the C and A domains, which are presumed to be inside the cell. This collection of these single-Cys BglF variants was expected to provide useful tools for establishing the operational parameters for our experimental approach and for investigating the actual membrane topology of BglF.Table IPrediction of transmembrane helices in BglF The numbers correspond to amino acid positions in the BglF protein.HMMTOPTMHMMPHDhtmTOPPREDMEMSATThis work102–121100–119108–125100–120100–124100–117142–164139–161144–161140–160140–164144–161171–190168–190170–190172–190173–190211–230205–227209–227211–231203–227208–230245–264234–256246–265245–265244–264246–263273–292260–282286–304287–307273–293274–296299–320287–309300–317325–346324–346325–345324–345355–374353–373355–375383–400381–400385–402381–401383–399407–424404–424406–422413–430429–448426–448427–446429–449429–449437–454No. of TMs1210711128 Open table in a new tab All together, 36 single-Cys BglF derivatives (with single cysteines between positions 97 and 480) were engineered as histidine-tagged proteins. The expression of only one mutant, T130C, was not detected after affinity purification or metabolic labeling with [35S]methionine (not shown). All other 35 single-Cys mutants were expressed in levels comparable with wild-type BglF, with the exception of variants with single-Cys at positions 141, 297, 313, 433 and 434, which where produced at slightly reduced levels (50-70% of wild-type level) (not shown). All single-Cys BglF variants were found in the membrane fraction after cell fractionation (not shown). The collection of single-Cys BglF mutants, whose expression was detected, was used to study the topology of the membrane C domain by cysteine accessibility analyses, as described below. Because the BglF active site, which phosphorylates the incoming β-glucosides and the BglG protein and also accepts the phosphate from P-BglG, is a cysteine residue (Cys-24), single-Cys BglF mutants are not active. Hence, we constructed a parallel set of di-Cys mutants, each containing Cys-24, residing in the B domain, plus one of the 35 cysteines described above to test the effect of the cysteine substitution in the C domain on BglF activity. The pattern of expression of the di-Cys variants was the same as that of the single-Cys mutants (not shown). The protocol of chemical modification by thiol-specific reagents was repeated with the di-Cys mutants. Most significantly, the results obtained with the two sets of mutants led to the same conclusions, as detailed below, indicating that a replacement of Cys-24 in the B domain by a serine does not affect the arrangement of the BglF derivatives in the membrane. Catalytic Activities of the Cys-replacement BglF Mutants—We assayed the ability of the di-Cys BglF mutants, containing the active site cysteine in the hydrophilic B domain, and a single cysteine in the C domain, to catalyze the various activities of BglF, i.e. sugar phosphotransfer and BglG phosphorylation and dephosphorylation. As a preliminary qualitative assay for sugar phosphotransfer, cells expressing the different di-Cys BglF variants were grown on MacConkey indicator plates containing the β-glucoside salicin or arbutin. Cells expressing functional BglF proteins catalyze uptake of salicin and arbutin; subsequent metabolism of these sugars leads to acidification of the medium and the appearance of red colonies. Cells expressing inactive BglF mutants form white colonies on MacConkey salicin/arbutin plates, whereas mutants with an intermediate activity grow as pink colonies. Of the 35 di-Cys mutants, only one, in position 432, formed white colonies on the indicator plates, and five others gave pink colonies (Table II). All other mutants grew as red colonies, indistinguishable from cells expressing wild-type BglF or the variant containing Cys-24 as a single cysteine (Cys-24 only). Hence, we concluded that all the di-Cys mutants, excluding one, retained at least some ability to transport and phosphorylate β-glucosides.Table IIThe activity of di-Cys BglF variants as sugar phosphotransferases and as BglG-negative regulators The background for all di-Cys BglF proteins was a BglF variant containing the active site cysteine as a sole cysteine (Cys-24 only).BglF mutantsβ-Glucosides phosphotransferaSugar phosphotransfer was studied in strain HMS174(DE3), which carries a cryptic bgl operon on its chromosomeRegulation of BglG activity in vivobThe ability of di-Cys BglF variants to negatively regulate BglG activity, depending on β-glucoside addition to the growth medium, was assayed in MA200-1, which carries a bgl′-lacZ transcriptional fusion on its chromosome and a defective bglF genePhenotype on MacConkey-arbutin platesc0.5% arbutin or 0.4% salicin were added to the growth medium when indicatedPhenotype on MacConkey-salicin platesc0.5% arbutin or 0.4% salicin were added to the growth medium when indicated,dTo assay salicin phosphotransfer, pZGM-B encoding the bglB gene, whose product hydrolyzes phosphorylated salicin, was introduced into the cells% Activity in pNPC assayeActivity was calculated as following: (A410 × 1000)/(A600 × 1 ml × 10 min). Wild type activity was defined as 100%. The values represent the average of at least three independent measurements. Standard deviation ranged from 0 to 16% activityPhenotype on MacConkey-lactose plates–Salicinc0.5% arbutin or 0.4% salicin were added to the growth medium when indicated+Salicinc0.5% arbutin or 0.4% salicin were added to the growth medium when indicatedWild typeRedRed100WhiteRedVector onlyWhiteWhite1RedRedC24SWhiteWhite2RedRedCys-lessWhiteWhite0RedRedCys-24 onlyRedRed101WhiteRedL97C + Cys-24RedRed95WhiteRedL125C + Cys-24RedRed87WhiteRedE136C + Cys-24RedRed93WhiteRedS138C + Cys-24RedRed96WhiteRedY141C + Cys-24RedRed79WhiteRedA163C + Cys-24RedRed90WhiteRedN192C + Cys-24RedRed125WhiteRedA196C + Cys-24RedRed101WhiteRedA198C + Cys-24RedRed100WhiteRedL201C + Cys-24RedRed94WhiteRedCys-227 + Cys-24RedRed93WhiteRedS240C + Cys-24RedRed86WhiteRedCys-251 + Cys-24RedRed90WhiteRedS271C + Cys-24PinkPink90WhiteRedA291C + Cys-24RedRed97WhiteRedF296C + Cys-24RedRed94WhiteRedW297C + Cys-24PinkPink74WhiteRedCys-313 + Cys-24RedRed85WhiteRedL320C + Cys-24RedRed103WhiteRedCys-346 + Cys-24RedRed93WhiteRedA350C + Cys-24RedRed90WhiteRedL364C + Cys-24RedRed99Whit" @default.
• W2036830304 created "2016-06-24" @default.
• W2036830304 creator A5004708642 @default.
• W2036830304 creator A5081773020 @default.
• W2036830304 date "2005-05-01" @default.
• W2036830304 modified "2023-09-29" @default.
• W2036830304 title "Dynamic Membrane Topology of the Escherichia coli β-Glucoside Transporter BglF" @default.
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