Title: The Inner Interhelix Loop 4–5 of the Melibiose Permease from Escherichia coli Takes Part in Conformational Changes after Sugar Binding
Abstract: Cytoplasmic loop 4–5 of the melibiose permease from Escherichia coli is essential for the process of Na+-sugar translocation (Abdel-Dayem, M., Basquin, C., Pourcher, T., Cordat, E., and Leblanc, G. (2003) J. Biol. Chem. 278, 1518–1524). In the present report, we analyze functional consequences of mutating each of the three acidic amino acids in this loop into cysteines. Among the mutants, only the E142C substitution impairs selectively Na+-sugar translocation. Because R141C has a similar defect, we investigated these two mutants in more detail. Liposomes containing purified mutated melibiose permease were adsorbed onto a solid supported lipid membrane, and transient electrical currents resulting from different substrate concentration jumps were recorded. The currents evoked by a melibiose concentration jump in the presence of Na+, previously assigned to an electrogenic conformational transition (Meyer-Lipp, K., Ganea, C., Pourcher, T., Leblanc, G., and Fendler, K. (2004) Biochemistry 43, 12606–12613), were much smaller for the two mutants than the corresponding signals in cysteineless MelB. Furthermore, in R141C the stimulating effect of melibiose on Na+ affinity was lost. Finally, whereas tryptophan fluorescence spectroscopy revealed impaired conformational changes upon melibiose binding in the mutants, fluorescence resonance energy transfer measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na+. These data suggest that: 1) loop 4–5 contributes to the coordinated interactions between the ion and sugar binding sites; 2) it participates in an electrogenic conformational transition after melibiose binding that is essential for the subsequent obligatory coupled translocation of substrates. A two-step mechanism for substrate translocation in the melibiose permease is suggested. Cytoplasmic loop 4–5 of the melibiose permease from Escherichia coli is essential for the process of Na+-sugar translocation (Abdel-Dayem, M., Basquin, C., Pourcher, T., Cordat, E., and Leblanc, G. (2003) J. Biol. Chem. 278, 1518–1524). In the present report, we analyze functional consequences of mutating each of the three acidic amino acids in this loop into cysteines. Among the mutants, only the E142C substitution impairs selectively Na+-sugar translocation. Because R141C has a similar defect, we investigated these two mutants in more detail. Liposomes containing purified mutated melibiose permease were adsorbed onto a solid supported lipid membrane, and transient electrical currents resulting from different substrate concentration jumps were recorded. The currents evoked by a melibiose concentration jump in the presence of Na+, previously assigned to an electrogenic conformational transition (Meyer-Lipp, K., Ganea, C., Pourcher, T., Leblanc, G., and Fendler, K. (2004) Biochemistry 43, 12606–12613), were much smaller for the two mutants than the corresponding signals in cysteineless MelB. Furthermore, in R141C the stimulating effect of melibiose on Na+ affinity was lost. Finally, whereas tryptophan fluorescence spectroscopy revealed impaired conformational changes upon melibiose binding in the mutants, fluorescence resonance energy transfer measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na+. These data suggest that: 1) loop 4–5 contributes to the coordinated interactions between the ion and sugar binding sites; 2) it participates in an electrogenic conformational transition after melibiose binding that is essential for the subsequent obligatory coupled translocation of substrates. A two-step mechanism for substrate translocation in the melibiose permease is suggested. Co-transporters catalyzing specifically the transfer of organic substrates and ions across membranes are of primary importance for the life of cells. One of these co-transporters belonging to the galactosides-pentoses-hexuronides transport family (1Poolman B. Knol J. van der Does C. Henderson P.J. Liang W.J. Leblanc G. Pourcher T. Mus-Veteau I. Mol. Microbiol. 1996; 19: 911-922Crossref PubMed Scopus (134) Google Scholar) is the melibiose permease (MelB) 4The abbreviations used are: MelB, melibiose permease; melibiose, 6-O-α-d-galactopyranosyl-d-glucose; α-NPG, p-nitrophenyl-α-d-6-galactopyranoside; RSO, right-side-out; ISO, inside-out; Dns2-S-Gal, 2′-(N-dansyl)aminoethyl-1-thio-β-d-galactopyranoside; FRET, fluorescence resonance energy transfer; SSM, solid supported membrane; MTSEA+, (2-aminoethyl)methanethiosulfonate hydrobromide; MPB, 3-(N-maleimidylpropionyl)biocytin; stilbene disulfonate, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate; NEM, N-ethylmaleimide; WT, wild type; Cys-less, cysteine less.4The abbreviations used are: MelB, melibiose permease; melibiose, 6-O-α-d-galactopyranosyl-d-glucose; α-NPG, p-nitrophenyl-α-d-6-galactopyranoside; RSO, right-side-out; ISO, inside-out; Dns2-S-Gal, 2′-(N-dansyl)aminoethyl-1-thio-β-d-galactopyranoside; FRET, fluorescence resonance energy transfer; SSM, solid supported membrane; MTSEA+, (2-aminoethyl)methanethiosulfonate hydrobromide; MPB, 3-(N-maleimidylpropionyl)biocytin; stilbene disulfonate, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate; NEM, N-ethylmaleimide; WT, wild type; Cys-less, cysteine less. of Escherichia coli. MelB functions as a secondary active co-transporter that links uphill solute transport (α-galactosides, such as 6-O-α-d-galactopyranosyl-d-glucose (melibiose), or β-galactosides, such as methyl-1-thio-β-galactopyranoside) to a downhill electrochemical ion gradient. It is most unusual in its ability to use either H+, Na+, or Li+ as coupling ion depending on which sugar is being transported (reviews in Refs. 1Poolman B. Knol J. van der Does C. Henderson P.J. Liang W.J. Leblanc G. Pourcher T. Mus-Veteau I. Mol. Microbiol. 1996; 19: 911-922Crossref PubMed Scopus (134) Google Scholar and 2Pourcher T. Bassilana M. Sarkar H.K. Kaback H.R. Leblanc G. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1990; 326: 411-423Crossref PubMed Scopus (48) Google Scholar). The binding of the cation enhances the affinity of the transporter for the co-transported sugar, and vice versa melibiose enhances the affinity of the transporter for Na+ (1Poolman B. Knol J. van der Does C. Henderson P.J. Liang W.J. Leblanc G. Pourcher T. Mus-Veteau I. Mol. Microbiol. 1996; 19: 911-922Crossref PubMed Scopus (134) Google Scholar, 2Pourcher T. Bassilana M. Sarkar H.K. Kaback H.R. Leblanc G. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1990; 326: 411-423Crossref PubMed Scopus (48) Google Scholar, 3Bassilana M. Pourcher T. Leblanc G. J. Biol. Chem. 1988; 263: 9663-9667Abstract Full Text PDF PubMed Google Scholar, 4Damiano-Forano E. Bassilana M. Leblanc G. J. Biol. Chem. 1986; 261: 6893-6899Abstract Full Text PDF PubMed Google Scholar, 5Ganea C. Pourcher T. Leblanc G. Fendler K. Biochemistry. 2001; 40: 13744-13752Crossref PubMed Scopus (35) Google Scholar). Consisting of 473 amino acids, this highly hydrophobic protein (70% apolar) has 12 transmembrane-spanning α-helices, organized as two asymmetric domains, each containing 6 helices, with its N and C termini facing the cytoplasm of the bacterium (6Hacksell I. Rigaud J.L. Purhonen P. Pourcher T. Hebert H. Leblanc G. EMBO J. 2002; 21: 3569-3574Crossref PubMed Scopus (41) Google Scholar, 7Gwizdek C. Leblanc G. Bassilana M. Biochemistry. 1997; 36: 8522-8529Crossref PubMed Scopus (39) Google Scholar, 8Botfield M.C. Naguchi K. Tsuchiya T. Wilson T.H. J. Biol. Chem. 1992; 267: 1818-1822Abstract Full Text PDF PubMed Google Scholar, 9Pourcher T. Bibi E. Kaback H.R. Leblanc G. Biochemistry. 1996; 35: 4161-4168Crossref PubMed Scopus (76) Google Scholar). While awaiting improved resolution of the MelB structure, other techniques are being used to understand better the functioning of this transporter and identify amino acids involved in substrate binding and/or translocation. For example, for MelB, Asp-19 (helix I), Asp-55 (helix II), Asp-59 (helix II), and Asp-124 (helix IV) have been shown to be essential for cation binding and Asn-58 (helix II) for cation recognition (10Pourcher T. Deckert M. Bassilana M. Leblanc G. Biochem. Biophys. Res. Commun. 1991; 178: 1176-1181Crossref PubMed Scopus (48) Google Scholar, 11Hama H. Wilson T.H. J. Biol. Chem. 1994; 269: 1063-1067Abstract Full Text PDF PubMed Google Scholar, 12Pourcher T. Zani M.L. Leblanc G. J. Biol. Chem. 1993; 268: 3209-3215Abstract Full Text PDF PubMed Google Scholar, 13Zani M.L. Pourcher T. Leblanc G. J. Biol. Chem. 1993; 268: 3216-3221Abstract Full Text PDF PubMed Google Scholar, 14Zani M.L. Pourcher T. Leblanc G. J. Biol. Chem. 1994; 269: 24883-24889Abstract Full Text PDF PubMed Google Scholar). Cysteine-scanning mutagenesis is now a widely used strategy to study systematically the structure and structure-function relationship of membrane proteins. It is currently applied to MelB using a fully functional active permease devoid of its four native cysteines (Cys-less permease) (15Weissborn A.C. Botfield M.C. Kuroda M. Tsuchiya T. Wilson T.H. Biochim. Biophys. Acta. 1997; 1329: 237-244Crossref PubMed Scopus (22) Google Scholar) as genetic background. Extensive cysteine-scanning mutagenesis studies concentrated thus far mainly on membrane domains of MelB. However, three observations led us to focus on the role of the highly charged cytoplasmic loop 4–5 for MelB symport activity. First, MelB substrates protect the protein cooperatively against proteolysis of loop 4–5 (7Gwizdek C. Leblanc G. Bassilana M. Biochemistry. 1997; 36: 8522-8529Crossref PubMed Scopus (39) Google Scholar). Second, active-site-directed labeling of the protein showed that the arginyl residue 141 of this same loop is a melibiose-protected target of labeling with a photoactivatable azidophenyl sugar analog (16Ambroise Y. Leblanc G. Rousseau B. Biochemistry. 2000; 39: 1338-1345Crossref PubMed Scopus (12) Google Scholar). From the latter study, it was concluded that loop 4–5 is either directly involved in Na+ and/or sugar binding or contributes indirectly to the coupling interaction between the two binding sites. Third, among the positively charged amino acids of loop 4–5 that were individually replaced by a cysteine, only R141C, although able to bind the substrates, showed defects in the translocation process (17Abdel-Dayem M. Basquin C. Pourcher T. Cordat E. Leblanc G. J. Biol. Chem. 2003; 278: 1518-1524Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) suggesting that loop 4–5 is close to the sugar binding site and may participate directly in co-substrate translocation. In the present study, we bring additional evidence for an important role of loop 4–5 for the functioning and cooperative behavior of MelB. We analyzed the effects of individual cysteine replacement of the three negatively charged residues; i.e. D137C, E140C, and E142C. Because among the mutants only E142C showed substrate translocation defects, this mutant, together with the previously characterized R141C mutant (17Abdel-Dayem M. Basquin C. Pourcher T. Cordat E. Leblanc G. J. Biol. Chem. 2003; 278: 1518-1524Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), was investigated in detail by combining kinetic, electrophysiological, and fluorescence spectroscopy approaches. In particular, we analyzed transient electrical currents recorded from R141C and E142C by using the solid supported membrane (SSM) technique. The SSM has already been used to monitor electrogenic events associated to partial steps of the Na+ melibiose symport reaction (5Ganea C. Pourcher T. Leblanc G. Fendler K. Biochemistry. 2001; 40: 13744-13752Crossref PubMed Scopus (35) Google Scholar, 18Meyer-Lipp K. Ganea C. Pourcher T. Leblanc G. Fendler K. Biochemistry. 2004; 43: 12606-12613Crossref PubMed Scopus (26) Google Scholar). In combination with spectroscopic evidence, the results suggest that loop 4–5 contributes to the coordinated interactions between the ion and sugar binding sites and participates in conformational changes after melibiose binding. Materials—Synthesis and labeling of p-nitrophenyl α-d-6-[3H]galactopyranoside (α-[3H]NPG) or 6-O-α-d-[3H]galactopyranosyl-d-glucose ([3H]melibiose), and synthesis of 2′-(N-dansyl)-aminoethyl-1-thio-β-d-galactopyranoside (Dns2-S-Gal) were carried out by Dr. B. Rousseau and Y. Ambroise (Département de Biologie Joliot Curie/CEA-Saclay, France). Highest purity grade reagents (KH2PO4, Sigma, 0.005% Na+ and KOH Merck, suprapur, 0.002% Na+) were used to prepare nominally Na+-free media (contaminating Na+ level of <20 μm as determined by atomic absorption spectroscopy). 3-(N-Maleimidylpropionyl)biocytin (biotin maleimide, MPB), 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate (stilbene disulfonate), and the streptavidin-alkaline phosphatase conjugate were purchased from Molecular Probes, Inc. (Eugene, OR). The methanethiosulfonate derivatives (MTS reagents) were from Toronto Research Chemicals, Inc. (Toronto, Canada). Escherichia coli lipid extract for the reconstitution of proteins and diphytanoylphosphatidylcholine for the lipid film forming solution were from Avanti Polar Lipids, Inc. (Pelham, AL). Bacterial Strains, Plasmids, and Site-directed Mutagenesis—A recombinant pK95ΔAHB plasmid with a cassette containing the melB gene (19Mus-Veteau I. Leblanc G. Biochemistry. 1996; 35: 12053-12060Crossref PubMed Scopus (31) Google Scholar) encoding a permease with a His6 tag at its C-terminal end (20Pourcher T. Leclercq S. Brandolin G. Leblanc G. Biochemistry. 1995; 34: 4412-4420Crossref PubMed Scopus (96) Google Scholar) and devoid of its four native cysteines (Cysless MelB (15Weissborn A.C. Botfield M.C. Kuroda M. Tsuchiya T. Wilson T.H. Biochim. Biophys. Acta. 1997; 1329: 237-244Crossref PubMed Scopus (22) Google Scholar)) was constructed by PCR and used as background for further permease engineering and as control. E. coli DW2-R (ΔmelB and ΔlacZY) (21Botfield M.C. Wilson T.H. J. Biol. Chem. 1988; 263: 12909-12915Abstract Full Text PDF PubMed Google Scholar) was transformed with the plasmid harboring the mutated MelB (Cys-less, D137C, R139C, E140C, R141C, or E142C, respectively). Freshly transformed cells were grown at 30 °C in M9 medium supplemented with glycerol (5 g/liter), casamino acids (0.2%), thiamine (0.5 mg/liter), and ampicillin (100 μg/ml) until an A600 of 1–1.2 was reached. The cells were washed and resuspended in 0.1 m potassium phosphate (KPi) at pH 7. Preparation of Membrane Vesicles—Right-side-out (RSO) membrane vesicles, prepared by an osmotic shock procedure (22Bassilana M. Damiano-Forano E. Leblanc G. Biochem. Biophys. Res. Commun. 1985; 129: 626-631Crossref PubMed Scopus (29) Google Scholar), were concentrated to 2 mg of protein/ml and equilibrated in a medium containing 0.1 m KPi (pH 7). Inside-out (ISO) membrane vesicles were generated using a French pressure cell as described previously (20Pourcher T. Leclercq S. Brandolin G. Leblanc G. Biochemistry. 1995; 34: 4412-4420Crossref PubMed Scopus (96) Google Scholar) and concentrated to ∼20 mg/ml in a medium containing 50 mm Tris-HCl (pH 8) and 50 mm NaCl (20Pourcher T. Leclercq S. Brandolin G. Leblanc G. Biochemistry. 1995; 34: 4412-4420Crossref PubMed Scopus (96) Google Scholar). Purification and Preparation of the Proteoliposomes—Mutated His-tagged MelB (Cys-less, D137C, R139C, R141C, or E142C) was purified from inverted membrane vesicles as described (20Pourcher T. Leclercq S. Brandolin G. Leblanc G. Biochemistry. 1995; 34: 4412-4420Crossref PubMed Scopus (96) Google Scholar). A chromatographic procedure combining the utilization of nickel-nitrilotriacetic acid (Qiagen, Germany) and ion exchange resin (Macro-Prep-High-Q anion exchange support, Bio-Rad) were used to prepare nearly pure MelB (generally >99% (20Pourcher T. Leclercq S. Brandolin G. Leblanc G. Biochemistry. 1995; 34: 4412-4420Crossref PubMed Scopus (96) Google Scholar)) solubilized in dodecylmaltoside (0.1%). MelB reconstitution into liposomes (protein/lipid ratio 1/5, w/w) (20Pourcher T. Leclercq S. Brandolin G. Leblanc G. Biochemistry. 1995; 34: 4412-4420Crossref PubMed Scopus (96) Google Scholar) was performed by removing the detergent with Bio-Beads SM-2 (Bio-Rad) (23Rigaud J.L. Paternostre M.T. Bluzat A. Biochemistry. 1988; 27: 2677-2688Crossref PubMed Scopus (229) Google Scholar). Proteoliposomes were submitted to repeated freeze/thaw/sonication wash cycles in nominally Na+-free, 0.1 m KPi (pH 7) to eliminate Na+ (contaminating level <10 μm as determined by flame photometry). Purity of the reconstituted MelB was assessed by silver-stained SDS-PAGE. Protein content was measured in the presence of SDS by a Lowry assay. Identification of Second Site Revertants—E. coli expressing E142C grew initially as pale rose colonies on 1% melibiose MacConkey agar plates. After 3–5 days of incubation at 37 °C, small red areas appeared that were picked and re-streaked for colony purification. After isolation of the plasmid DNA, mutations responsible for fermentation recovery were identified by sequencing. Sugar Transport in Cells—Freshly grown cells were concentrated to 2 mg of protein/ml in 0.1 m KPi (pH 7). The time course of transport of [3H]melibiose (0.4 mm, 20 mCi/mmol) in the presence or absence of NaCl or LiCl at a concentration of 10 mm was monitored at 22 °C by a rapid filtration procedure (17Abdel-Dayem M. Basquin C. Pourcher T. Cordat E. Leblanc G. J. Biol. Chem. 2003; 278: 1518-1524Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Variation of transport rate and extent never exceeded 15% from batch to batch. Western Blot Analysis—Western blot analysis was applied to samples containing protein solubilized from RSO membrane vesicles (∼20 μg) and probed with a mouse anti-C-terminal MelB and an anti-mouse secondary antibody (7Gwizdek C. Leblanc G. Bassilana M. Biochemistry. 1997; 36: 8522-8529Crossref PubMed Scopus (39) Google Scholar). Results were expressed as the percentage of the signal recorded from membranes carrying the Cys-less permease. Binding Assays—α-[3H]NPG binding to RSO membrane vesicles (20 mg/ml) and determination of binding constants were assessed at room temperature by a flow dialysis procedure as described previously (4Damiano-Forano E. Bassilana M. Leblanc G. J. Biol. Chem. 1986; 261: 6893-6899Abstract Full Text PDF PubMed Google Scholar). Entrance Counterflow Activity—Counterflow activity was assessed in RSO membrane vesicles (20 mg/ml) at room temperature as described (17Abdel-Dayem M. Basquin C. Pourcher T. Cordat E. Leblanc G. J. Biol. Chem. 2003; 278: 1518-1524Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Solid Supported Membrane Set-up and Measuring Procedure—Proteoliposomes containing Cys-less, R141C, or E142C MelB were adsorbed to the SSM (5Ganea C. Pourcher T. Leblanc G. Fendler K. Biochemistry. 2001; 40: 13744-13752Crossref PubMed Scopus (35) Google Scholar, 24Pintschovius J. Fendler K. Bamberg E. Biophys. J. 1999; 76: 827-836Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Activating and non-activating solutions for SSM measurements contained 0.1 m KPi (pH 7) plus NaCl/KCl and melibiose/glucose at concentrations as indicated in the figure legends. Transient currents upon addition of one or both of the co-substrates were recorded. The solution exchange protocols were as described (5Ganea C. Pourcher T. Leblanc G. Fendler K. Biochemistry. 2001; 40: 13744-13752Crossref PubMed Scopus (35) Google Scholar). All experiments were carried out at room temperature (22 °C). Fluorescence Assays—An LS 50 fluorometer (PerkinElmer Life Sciences) was used to measure the Na+-dependent fluorescence resonance energy transfer (FRET) signals (λex = 297 nm, bandpass 5 nm, λem = 410–540 nm) arising from proteoliposomes (20 μg/ml) incubated in the presence of 10 μm of the sugar fluorescent analog Dns2-S-Gal (25Maehrel C. Cordat E. Mus-Veteau I. Leblanc G. J. Biol. Chem. 1998; 273: 33192-33197Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The specific FRET signals were normalized for variation (<20%) in the amount of permeases in the different proteoliposome preparations according to a previous study (26Cordat E. Mus-Veteau I. Leblanc G. J. Biol. Chem. 1998; 273: 33198-33202Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Intrinsic tryptophan fluorescence (λex = 297 nm, bandpass 5 nm, λem = 310–380 nm) arising from proteoliposomes (20 μg/ml) was recorded as described (27Mus-Veteau I. Pourcher T. Leblanc G. Biochemistry. 1995; 34: 6775-6783Crossref PubMed Scopus (37) Google Scholar). Orientation of the Protein in the Liposome—Liposome suspensions containing purified Cys-less or single cysteine R139C (∼0.25 mg of protein/ml, in 0.1 m KPi, pH 7) were supplemented with 10 μm MPB and incubated for 5 min at room temperature. MPB was added from a stock prepared in Me2SO. The concentration of Me2SO in the labeling medium did not exceed 1% (v/v). Some samples were afterward subjected to three cycles of freeze-thaw sonication in the presence of MPB. Where indicated, samples were pretreated with 200 μm of the membrane-impermeable thiolstilbene disulfonate for 10 min at room temperature. Stilbene disulfonate was washed away by three cycles of centrifugation with 0.1 m KPi, pH 7. As negative control, the Cys-less transporter was incubated with varying concentrations of MPB (0–1000 μm) and subjected to three cycles of freeze-thaw sonication. The biotin maleimide-labeling reaction was stopped by the addition of 10 μm dithiothreitol to the samples. All samples were solubilized in 1% SDS and subjected to SDS-PAGE (10%) and Western Blot analysis. A streptavidin-alkaline phosphatase conjugate was used for the specific reaction with biotin maleimide and a MelB-specific monoclonal antibody (21E4, Biocytex, Marseille, France), coupled directly to an alkaline phosphatase (Davids Biotechnologie, Regensburg, Germany), for the determination of the amount of protein. Both antibodies were used in a 1:1000 dilution. The alkaline phosphatase conjugate substrate Kit (Bio-Rad) was used to visualize the bands on the membrane. The three acidic residues of loop 4–5 of MelB (Asp-137, Glu-140, and Glu-142) and the positively charged residues Arg-139 or Arg-141 (17Abdel-Dayem M. Basquin C. Pourcher T. Cordat E. Leblanc G. J. Biol. Chem. 2003; 278: 1518-1524Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) were individually replaced by a cysteine using the Cys-less MelB sequence as genetic background. The Cys-less MelB served also as control throughout this study as its functional properties are similar to those reported for the wild-type MelB (15Weissborn A.C. Botfield M.C. Kuroda M. Tsuchiya T. Wilson T.H. Biochim. Biophys. Acta. 1997; 1329: 237-244Crossref PubMed Scopus (22) Google Scholar, 17Abdel-Dayem M. Basquin C. Pourcher T. Cordat E. Leblanc G. J. Biol. Chem. 2003; 278: 1518-1524Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Functional Properties of D137C, E140C, and E142—Initial characterization of D137C, E140C, and E142C MelB mutants included: 1) the measurement of the time course of melibiose accumulation in bacteria in the presence or absence of saturating concentrations of activating Na+ or Li+ (10 mm) and 2) the estimation of the expression level of the mutated transporter. Although initial rate and sugar accumulation at equilibrium in the presence of Na+ (10 min) by D137C or E140C cells were 0.9 or 0.45 times the values measured in Cys-less MelB, the level of E142C sugar accumulation was reduced by a factor of 10 (Fig. 1A). Similar to the wild-type, melibiose transport activity was stimulated by Na+ and Li+ both in the Cys-less mutant and in D137C, E140C, and E142C (Fig. 1A). Strikingly, the residual transport observed in E142C was completely abolished in the presence of 0.2 mm N-ethylmaleimide (NEM), whereas that of D137C or E140C was not sensitive to the sulfhydryl reagent (data not shown). Comparison of the expression levels of Cys-less MelB and the mutants suggests that the reduced transport activity of D137C and E140C MelB can be satisfactorily accounted for by the reduction of the expression level of the two mutants (∼70 and ∼60% of Cys-less, respectively, not shown). In contrast, the permease content of E142C in membranes was proportionally too high (∼40%) to explain the large drop in cell transport activity. In view of these results, the E142C mutant was further characterized, and its properties were compared with those of the Cys-less and the R141C mutants. Measurement of sugar binding affinity in RSO membrane vesicles using the high affinity radiolabeled sugar analog α-[3H]NPG (4Damiano-Forano E. Bassilana M. Leblanc G. J. Biol. Chem. 1986; 261: 6893-6899Abstract Full Text PDF PubMed Google Scholar) as a ligand revealed that E142C retained the ability to bind the sugar in a Na+-dependent manner. However, the affinity for the sugar analog of E142C was lowered by a factor of 3–4 as compared with that of Cys-less MelB (apparent KD (Cys-less) = 0.6 μm, apparent KD (E142C) = 3.4 μm, data not shown). This drop in sugar affinity was similar to that observed in R141C (17Abdel-Dayem M. Basquin C. Pourcher T. Cordat E. Leblanc G. J. Biol. Chem. 2003; 278: 1518-1524Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In contrast, the Na+ activation constants for α-NPG binding (KNa) of E142C and Cys-less were comparable (both had a KNa of 0.32 mm, data not shown). Although NEM inhibited the residual transport in E142C, the sulfhydryl reagent added before or after addition of any substrate to the vesicles had no detectable effect on its sugar-binding properties (data not shown). On the other hand, NEM had no effect on the substrate binding or translocation properties of the Cys-less mutant (data not shown). The sugar translocation properties of MelB can be assessed by measuring entrance counterflow in de-energized RSO membrane vesicles (17Abdel-Dayem M. Basquin C. Pourcher T. Cordat E. Leblanc G. J. Biol. Chem. 2003; 278: 1518-1524Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The early transient influx of labeled substrate (overshoot) is tightly coupled to the downhill efflux of internal unlabeled sugar occurring when RSO membrane vesicles, pre-loaded with a sugar at high concentration, are diluted in a medium containing a lower sugar concentration (2Pourcher T. Bassilana M. Sarkar H.K. Kaback H.R. Leblanc G. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1990; 326: 411-423Crossref PubMed Scopus (48) Google Scholar, 28Kaback H.R. Sahin-Toth M. Weinglass A.B. Nat. Rev. Mol. Cell Biol. 2001; 2: 610-620Crossref PubMed Scopus (249) Google Scholar). The radiolabeled sugar influx is primarily associated to shuttling of the loaded ternary complex (MelB-ion-sugar) across the membrane and does not include a contribution of the empty carrier. The peak of the transient uptake of [3H]melibiose observed after 1 min in Cys-less MelB was absent in E142C (Fig. 1B). Instead, the intravesicular level of radioactive melibiose in E142C steadily increased until a plateau value was reached. This influx of labeled sugar was significantly inhibited by NEM (data not shown). Summarizing, the defect of melibiose transport function of E142C included both a small reduction of sugar affinity and an impaired sugar translocation process. As will be further emphasized in the discussion, it is striking that mutagenesis of the neighboring E142C and R141C residues (17Abdel-Dayem M. Basquin C. Pourcher T. Cordat E. Leblanc G. J. Biol. Chem. 2003; 278: 1518-1524Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) gave rise to similar defects of the MelB translocation mechanism. It is finally worth mentioning that we isolated a second-site revertant of E142C with an additional mutation on I22S that is positioned on the inner half of helix I (E142C/I22S). Despite a permease expression level only slightly higher than that of E142C (∼50% versus ∼40%, respectively, data not shown), the revertant exhibited a Na+-stimulated melibiose transport activity five times higher than that of E142C (Fig. 1C) and a higher α-NPG affinity (apparent KD = 2.1 μm versus 3.4 μm, data not shown). The fact that a substitution of an apolar residue (isoleucine) by a polar residue (serine) located on helix I was able to compensate the loss of the negative charge at position 142 suggests first that helix I and loop 4–5 might be close to each other. Secondly, while this negative charge is not absolutely required, the need for a local polar environment at its level is important for the transport function and is party satisfied by introduction of the Ile to Ser mutation at position 22. Charge Translocation of R141C and E142C—Further insights into the R141C (17Abdel-Dayem M. Basquin C. Pourcher T. Cordat E. Leblanc G. J. Biol. Chem. 2003; 278: 1518-1524Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) and E142C defects were gained by comparing transient electrical currents recorded from liposomes containing purified mutated MelB, which were adsorbed onto the SSM and submitted to MelB co-substrate concentration jumps (5Ganea C. Pourcher T. Leblanc G. Fendler K. Biochemistry. 2001; 40: 13744-13752Crossref PubMed Scopus (35) Google Scholar). Previous studies have shown that the transient signals recorded from wild-type MelB containing proteoliposomes include either a single fast decaying component (in the range of 10 ms) or a combination of a fast and a slow decaying component (range of 100 ms) depending on the composition of the imposed substrate concentration jump (5Ganea C. Pourcher T. Leblanc G. Fendler K. Biochemistry. 2001; 40: 13744-13752Crossref PubMed Scopus (35) Google Scholar). Whereas the fast component was assigned to intraprotein charge transfers associated to or triggered by the binding of either co-substrate, th
Publication Year: 2006
Publication Date: 2006-09-01
Language: en
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