Cytoplasmic Loop Connecting Helices IV and V of the Melibiose Permease from Escherichia coli Is Involved in the Process of Na+-coupled Sugar Translocation*

Manal Abdel DayemDagger, Cécile Basquin, Thierry Pourcher, Emmanuelle Cordat, and Gérard Leblanc§

From the Laboratoire de Physiologie des Membranes Cellulaires, Université de Nice Sophia-Antipolis and CNRS UMR 6078, Commissariat à l'Energie Atomique (LRC-CEA 16V), Villefranche sur mer, 06230 France

Received for publication, October 1, 2002, and in revised form, November 4, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous photolabeling and limited proteolysis studies suggested that one of the four basic residues (Arg-141) of the N-terminal cytoplasmic loop connecting helices IV and V (loop 4-5) of the melibiose permease (MelB) from Escherichia coli has a potential role in its symport function (Ambroise, Y., Leblanc, G., and Rousseau, B. (2000) Biochemistry 39, 1338-1345). A mutagenesis study of Arg-141 and of the other three basic residues of loop 4-5 was undertaken to further examine this hypothesis. Cys replacement analysis indicated that Arg-141 and Arg-149, but not Lys-138 and Arg-139, are essential for MelB transport activity. Replacement of Arg-141 by neutral residues (Cys or Gln) inactivated transport and energy-independent carrier-mediated flows of substrates (counterflow, efflux), whereas it had a limited effect on co-substrate binding. R141C sugar transport was partially rescued on reintroducing a positive charge with a charged and permeant thiol reagent. Whereas R149C was completely inactive, R149K and R149Q remained functional. Strikingly, introduction of an additional mutation in the C-terminal helix X (Gly for Val-343) of R149C restored sugar transport. Impermeant thiol reagents inhibited R149C/V343G transport activity in right-side-out membrane vesicles and prevented sugar binding in a sugar-protected manner. All these data suggest that MelB loop 4-5 is close to the sugar binding site and that the charged residue Arg-141 is involved in the reaction of co-substrate translocation or substrate release in the inner compartment.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The melibiose permease (MelB)1 of Escherichia coli is a membrane-bound ion-coupled sugar co-transporter or symporter that drives cell accumulation of alpha -galactosides (melibiose, raffinose) or beta -galactosides (methyl-1-thio-beta -galactopyranoside (TMG)) using a favorable electrochemical potential gradient for Na+, Li+, or H+ (1-3). The MelB symporter (473 amino acids, 53 kDa) has 12 helical membrane domains (4-8). A recombinant transporter bearing a His6 tag (Mel-His6 permease) can be purified in large amounts and exhibits ion-dependent sugar binding and transport properties similar to MelB in its native environment once reconstituted in artificial lipid membranes (9-11). Recently, two-dimensional crystallization of Mel-His6 permease was achieved, and a projection map at 8 Å resolution was derived by electron crystallography (12). The projection map suggests an asymmetric protein unit hosting 12 potential transmembrane alpha -helices that are distributed in two domains lining a central and curve-shaped cleft.

Na+ and melibiose are transported in a 1:1 ratio, and their order of binding to MelB at the outer surface (Na+ first) and release in the inner compartment (Na+ last) are sequential (13). The membrane potential enhances active Na+-coupled sugar transport by increasing the rate of Na+ dissociation in the cytoplasm, which is otherwise slow and rate-limiting for MelB turnover (see review, Ref. 3). Cooperative changes in MelB conformation upon substrate binding were inferred from the analysis of the intrinsic fluorescence of MelB and by using fluorescence resonance energy transfer spectroscopy (FRET) with a fluorescent sugar analog (10, 14). Complementary evidence came from the finding that MelB substrates afford a cooperative protection against proteolysis of the highly charged cytoplasmic loop 4-5 connecting helices IV and V of MelB (7).

Molecular biology, biochemical, and spectroscopic approaches have been extensively used to collect information on the functional organization of the transporter. This organization involves the presence of a potential coordination network for the cation recognition and coupling mechanism, including four aspartic acid residues distributed in the inner halves of helices I (Asp-19), II (Asp-55 and Asp-59), and IV (Asp-124) of the N-terminal hydrophobic domain of MelB (6, 15-18). Analysis of the properties of second-site suppressor mutants supports the hypothesis of a proximity between the three N-terminal helices (19, 20). Extensive Cys-scanning mutagenesis of residues of membrane domains combined with chemical modifications of MelB with impermeant thiol reagents or with second-site mutation analysis has been used to identify sectors of given membrane domains facing the co-substrate pathway. They also suggest proximity between helices I, II, IV, VII, X, and XI (see Ref. 20 and references therein).

To date, most of the previous analyses focused on the membrane domains of MelB. However, few reports have already pointed out a possible functional implication of cytoplasmic loop 10-11 connecting helices X and XI (3, 21-23). In addition, two other observations suggested that the cytoplasmic loop connecting helices IV and V (loop 4-5) might also be important for MelB function (see Fig. 1). The first one showed that the arginyl residue Arg-141 of loop 4-5 is a melibiose-protected site of MelB labeling by a photoactivable azido phenyl sugar analog (24). The other one indicated that Arg-141 is part of one of the two trypsin cleavage targets in this loop 4-5 that are cooperatively protected against protease attack in the presence of the physiological substrates (Na+ + melibiose > Na+ melibiose) (7). To elucidate the role of Arg-141 and loop 4-5, we analyzed the functional consequences of replacing Arg-141 by different residues (Cys, Lys, Gln) and compared them with the effects produced by the mutagenesis of the three other basic residues of this loop (Lys-138, Arg-139, and Arg-149). Complementary information was obtained by reacting the newly introduced Cys residues with methanethiosulfonate derivatives (25) and isolation of second-site suppressor mutants. The obtained data suggest that the cytoplasmic loop 4-5 of MelB is close to the sugar binding site and may participate directly in co-substrate translocation by MelB.


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Fig. 1.   Secondary structure model of melibiose permease (5). Helices are represented by rectangles, a number of which are labeled with roman letters. Top and bottom numbers in the rectangles correspond to the first and last residues in the helices. The zoomed-in area shows the amino acid composition of cytoplasmic loop 4-5 connecting membrane-spanning segments IV and V. Shadowed letters indicate the positively charged residues subjected to individual cysteine replacement and lettered according to their position on the MelB sequence. The second-site mutation present in helix X (V343G) of the revertant R149C/V343G described below is indicated in bold face.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Materials-- Synthesis and labeling of p-nitrophenyl alpha -D-6-[3H]galactopyranoside (alpha -[3H]NPG) or 6-O-alpha -D-[3H]galactopyranosyl-D-glucose and synthesis of the fluorescent sugar analog (Dns2-S-Gal) were carried out by Dr. B. Rousseau and Y. Ambroise (Département de Biologie Joliot Curie/CEA-Saclay, France). [14C]TMG was from Isotopchim (France). The methanethiosulfonate derivative reagents (MTSEA+, MTSET+) were purchased from Toronto Research Chemicals, Inc. Stock solutions (0.2-0.5 M) were prepared in distilled water, stored in the frozen state, and used immediately after thawing. Stock solutions of N-ethylmaleimide (Sigma) were prepared at 0.2 M in ethanol.

Bacterial Strains and Cell Growth-- E. coli DW2-R, a Rec A- derivative of strain DW2 (Delta melB Delta lacZY), was transformed with the given plasmids (9). Freshly transformed cells were grown at 30 °C in M9 medium supplemented with glycerol (0.5 g/liter) and casamino acids (0.2%) as the carbon source, thiamine (0.5 mg/ml), and ampicillin (100 µg/ml) until an A600 of 1-1.2 was reached. Cells were then washed and resuspended in 0.1 M potassium phosphate buffer (pH 7).

Plasmid, Site-directed Mutagenesis, and DNA Sequencing-- A recombinant pK95Delta AHB plasmid with a cassette containing the melB gene (9) encoding a permease devoid of its four native cysteine (C-less MelB) was constructed with the appropriate mutagenesis primers and subsequently used for control experiments or for permease engineering. This C-less carrier displayed a valine instead of Cys-235 and a serine instead of Cys-110, Cys-310, and Cys-364 (26). Individual Cys replacements were engineered by polymerase chain reactions using the appropriate primers (27-30 nucleotides, Genosys) and the recombinant plasmid harboring the C-less carrier melB gene as a matrix. Plasmids were isolated using the QIAprep Spin Miniprep Kit (Qiagen). Sequencing was performed using 35S-labeled dATP with the sequencing kit (Amersham Biosciences).

Preparation of Membrane Vesicles-- Right-side-out (RSO) membrane vesicles prepared by an osmotic shock procedure as described (2) were concentrated to 2 mg of protein/ml and equilibrated in a medium containing 0.1 M potassium phosphate (pH 7) lacking NaCl (<20 µM).

Sugar Transport in Cells or RSO Membrane Vesicles-- Freshly grown cells were concentrated to 2 mg of protein/ml in a 0.1 M potassium phosphate (pH 7) medium that contained NaCl at a final concentration lower than 20 µM. H+-coupled sugar transport was assayed in this medium by adding either [3H]melibiose (20 mCi/mmol) or [14C]TMG (3 mCi/mmol) at a final concentration of 0.4 or 0.8 mM. The assay was stopped after the given times by diluting and immediate filtration of the samples using glass fiber filters (7). When Na+- or Li+-dependent sugar transport was measured, NaCl or LiCl was added at a final concentration of 10 mM just before the addition of the radioactive substrates. Transport in RSO membrane vesicles was assayed by a similar procedure except for the presence of reduced phenazine methosulfate as an energy donor (2). The radioactivity retained on the filters was counted in a Packard scintillation counter.

Entrance Counterflow Activity-- RSO membrane vesicles (20 mg/ml) were previously loaded with 30 mM melibiose for 12 h at 4 °C. 2-µl aliquots of sugar-loaded vesicles were diluted 100-fold in a buffer containing [3H]melibiose at a concentration of 0.1 mM (40 mCi/mmol). The extra-vesicular unlabeled sugar present in the membrane aliquots raised the final concentration of sugar of the diluting medium to 0.4 mM (10 mCi/mmol). The intra-vesicular accumulation of radioactivity was terminated at the given times by a dilution/filtration procedure similar to that used for transport assays.

Binding Assays-- alpha -[3H]NPG binding to RSO membrane vesicles was assessed by a flow dialysis procedure as described in Damiano-Forano et al. (13). RSO membrane vesicle suspensions (10-20 mg/ml) were initially equilibrated with alpha -[3H]NPG (0.8 mCi/mmol), and the medium NPG concentration was varied between 0.1 and 10 µM by the stepwise addition of unlabeled ligand. The maximal number of binding sites (Bmax) and apparent binding constant (KD) were calculated graphically from Scatchard plots of the data. The activation constant for NPG binding by Na+ (KNa) was determined using plots of the apparent KD values for NPG as a function of the reciprocal concentration of sodium chloride in the range of 0.1-10 mM. Melibiose inhibitory constants (Ki) were estimated by progressive displacement of bound alpha -[3H]NPG (0.8 mCi/mmol, 1 µM) by stepwise addition of sugar (0.5-20 mM). These Ki values were used to calculate the melibiose binding constants (KD) (27).

Western Blot Analysis and Protein Determinations-- Samples containing solubilized RSO membrane protein (~20 µg) were subjected to SDS, 12% polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The electroblotted proteins were probed with a mouse anti-C-terminal MelB antibody followed by incubation with a horseradish peroxidase-linked sheep antibody and visualized by enhanced chemiluminescence (7). Results were expressed as percent of the signal recorded from membranes carrying the Cys-less permease (0.4-0.6 nmol/mg of protein, see Table I). Cell or membrane protein was estimated by a modified Folin procedure, with bovine serum albumin as a standard.

Fluorescence Assays-- A LS 50 PerkinElmer fluorometer was used to measure the Na+-dependent FRET signals (Eex, 297 ± 5 nm; E<UP><SUB>em</SUB><SUP>max</SUP></UP>, 465-475 ± 5 nm) arising from RSO membrane vesicles (100 µg of protein/ml) incubated in the presence of the sugar fluorescent analog Dns2-S-Gal at a final concentration of 10 µM (14).

Identification of R149C/V343G Second-site Revertants-- A strategy developed by Ding and Wilson was used (28). Briefly, DW2-R-melA+ cells expressing R149C permease grew initially as white colonies on 1% melibiose MacConkey agar plates (Difco). After 3-5 days incubation at 37 °C, small red areas appeared and were picked and re-streaked for colony purification. After isolation of the plasmid DNA, mutations responsible for fermentation recovery were identified by sequencing the entire gene using primers annealing at about 250-bp intervals.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transport and Binding Properties of K138C, R139C, R141C, or R149C Permeases-- Throughout this study the C-less MelB permease retaining a wild-type transport phenotype was used as control (see Fig. 2, Table I, and Ref. 26). The functional consequences of individual replacements of each four positively charged residues of loop 4-5 by a cysteine was assessed by measuring Na+-dependent [3H]melibiose accumulation in E. coli DW2-R cells expressing each mutant. Fig. 2A shows that K138C and R139C cells accumulated [3H]melibiose in 10 min to an extent close to or higher than C-less MelB cells. Western blot analysis suggested that the difference in transport could be accounted for by variation of the cell membrane permease contents (R139C > C-less MelB > K138C, data not shown). The sugar (melibiose, TMG) and ionic selectivity profiles (Na+, Li+, or H+) of K138C and R139C permeases were similar to that of C-less MelB (data not shown). In contrast, neither R141C nor R149C permeases catalyzed significant cell accumulation of melibiose (Fig. 2A) or of TMG (data not shown). Because R141C and R149C membrane permease contents were 65 and 74% that of C-less MelB membrane, respectively (Fig. 2B), these two mutants must have a catalytic defect.


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Fig. 2.   Cell sugar transport by K138C, R139C, R141C, or R149C MelB mutants. A, [3H]melibiose transport by E. coli DW2-R cells transformed with given recombinant plasmids. Control experiments were conducted using the C-less MelB permease. Cell transport was assayed using cells (2 mg of protein/ml) equilibrated in 0.1 M potassium phosphate (pH 7). Transport was started immediately after the addition of NaCl and [3H]melibiose (20 mCi/mmol) at final concentrations of 10 and 0.4 mM, respectively, and terminated at the indicated time points by a rapid dilution followed by immediate filtration. B, permease contents of R141C and R149C. Western blot analysis of the permease expression levels in RSO membrane vesicles was carried out with anti-C-terminal antibody as described under "Experimental Procedures."

                              
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Table I
NPG binding constants of the C-less permease, of the permease carrying different substitutions at position 141 or 149, and of the second-site revertant R149C/V343G
All RSO membrane vesicles harboring the C-less or MelB mutants were prepared and concentrated (15-25 mg of protein/ml) in Na--free, 0.1 M potassium phosphate buffer (pH 7) containing carbonyl cyanide m-chlorophenylhydrazone (5 µM) and monensin (0.75 µM) as described in the Fig. 3 legend. alpha -[3H]NPG binding was assayed in a flow dialysis cell as described in Damiano-Forano et al. (13). alpha -[3H]NPG binding was determined in vesicle suspensions initially equilibrated with alpha -[3H]NPG (0.8 mCi/mmol), and the medium NPG concentration was varied in the range of 0.1-10 µM by the stepwise addition of unlabeled ligand. The maximal number of binding sites (Bmax) and apparent NPG binding constant (KD) were calculated graphically from Scatchard plots of the data. The activation constant for NPG binding by Na+ (KNa) was determined from plots of the apparent KD for NPG as a function of the reciprocal concentration of sodium chloride in the range of 0.1-10 mM. Values are the means of 3 experiments, and S.E. were generally <20%. NS, not significant.

To assess more precisely the catalytic defect(s) of R141C and R149C permeases, their Na+-dependent sugar binding activity was measured in RSO membrane vesicles using the high affinity radiolabeled sugar analog alpha -[3H]NPG as a ligand (13). The upper curve in Fig. 3A illustrates typical binding variations recorded from de-energized C-less MelB membrane vesicles. The drop of free alpha -[3H]NPG concentration observed on adding NaCl (20 mM) reflects enhanced alpha -[3H]NPG binding to C-less MelB membranes, whereas the subsequent rise occurring upon the addition of unlabeled NPG in excess (250 µM) was due to the release of bound ligand (upper curve). Fig. 3A also shows that R141C membranes retained Na+-dependent NPG binding activity (middle curve), whereas R149C membranes did not (lower curve), even when the NaCl concentration was raised up to 1 M (data not shown). Additional binding experiments indicated that R141C permease had at most 3-4 times less affinity for alpha -[3H]NPG (KD, Table I) or for melibiose (2 mM instead of 0.6 mM, not shown) than C-less permease. In contrast, no significant change in activation of sugar binding to the two mutants by sodium was observed (KNa, Table I).


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Fig. 3.   Na+-dependent alpha -[3H]NPG binding and FRET signal recorded from C-less MelB, R141C, or R149C RSO membrane vesicles. A, Na+-dependent alpha -[3H]NPG binding to RSO membrane vesicles harboring each permease was assayed by means of a flow dialysis technique (13). RSO membrane vesicles concentrated to about 10 mg of protein/ml (C-less MelB) or 15-20 mg of protein/ml (R141C, R149C) were equilibrated at 20 °C in Na+-free potassium phosphate (100 mM, pH 7) buffer containing alpha -[3H]NPG (0.8 mCi/mmol, 1 µM) carbonyl cyanide m-chlorophenylhydrazone (ClCCP) (5 µM) and monensin (0.75 µM) in the upper chamber of the dialysis cell. When indicated by arrows, NaCl and unlabeled alpha -NPG were successively added at a final concentration of 20 mM or 250 µM, respectively. Ordinate, radioactivity (cpm) collected from the lower chamber of the dialysis cell used as a reporter of the concentration of free alpha -[3H]NPG in the membrane suspension. B, Na+-dependent variation of the FRET signal recorded from C-less MelB, R141C, or R149C RSO membrane vesicles incubated in the presence of the fluorescent sugar analog Dns2-S-Gal. De-energized RSO membrane vesicles (100 µg of protein/ml) were incubated with Dns2-S-Gal (10 µM) and excited at 297 ± 5 nm (14). The FRET signal (average 10 scans) was recorded before and after the addition of NaCl (20 mM) to the suspension, and the Na+-dependent signal was calculated by subtracting the spectra.

FRET spectroscopy of RSO membrane vesicles incubated in the presence of beta -galactoside fluorescent analog Dns2-S-Gal provides the means to assess ion-induced structural changes of MelB and related variation of the sugar binding site microenvironment (14). The specific Na+-induced, tryptophan-mediated fluorescence signal (Emax at 465 nm) recorded from C-less MelB membrane vesicles excited at 297 ± 5 nm (upper spectrum, Fig. 3B) was similar to that previously recorded for wild-type MelB membrane vesicles. More interesting, R141C permease retained comparable Na+-dependent FRET signal (middle spectrum), indicating that Na+ ions still induced a cooperative modification of the sugar binding site structure in this mutant. In contrast, no significant FRET signal was recorded from R149C membranes (lower spectrum). From all these data, we conclude that the R141C mutation introduces a defect in the transport cycle at a step that follows co-substrate binding, whereas the R149C mutation directly impairs sugar binding.

A Positive Charge Is Required at Position 141 for Co-substrate Translocation-- Entrance counterflow is an experimental assay where a very large increase in inward movement of radioactive sugar can be triggered by diluting de-energized RSO membrane vesicles loaded with unlabeled sugar (30 mM) into a medium containing labeled sugar at 0.4 mM. It is accepted that the influx of the labeled substrate during the initial phase of the trans-stimulation process is primarily associated to shuttling of the loaded ternary complex (MelB-ion-sugar) across the membrane and does not include contribution of the empty carrier (3, 29). A transient uptake of [3H]melibiose with a peak at about 1 min was observed in control C-less MelB membranes (Fig. 4A) but not in R141C vesicles. In addition, downhill efflux of [3H]melibiose from sugar-loaded R141C membrane vesicles was completely inhibited (data not shown). All these data can be explained assuming an impaired reorientation of the loaded R141C carrier or sugar release and probably also sugar binding at the cytoplasmic surface.


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Fig. 4.   Restoration of MelB entrance counterflow or transport in R141C RSO membrane vesicles with the permeant thiol modifier MTSEA+. A, entrance counterflow activity. R141C (squares) or C-less (circles) RSO membrane vesicles were concentrated to about 20 mg of protein/ml in 0.1 M potassium phosphate buffer (pH 7) containing NaCl (10 mM), monensin (0.75 µM), and carbonyl cyanide m-chlorophenylhydrazone (ClCCP) (5 µM) and equilibrated overnight at 4 °C in the presence of melibiose at a final concentration of 30 mM. Counterflow was assayed by diluting the loaded membrane vesicles 200-fold into the same saline medium containing [3H]melibiose (20 mCi/mmol) at a final concentration of 0.4 mM. At the indicated time, the reactions were terminated by rapid filtration. When tested, MTSEA+ was added to the loaded vesicle suspension at a final concentration of 1 mM 5 min before dilution (closed squares). B, [3H]melibiose accumulation. Active transport was assayed on well aerated aliquots of RSO membrane vesicles harboring C-less (open circles) R141C (open squares) permeases (50 µl, 2 mg protein/ml) prepared in 0.1 M potassium phosphate buffer (pH 7) containing NaCl (10 mM) and reduced phenazine methosulfate (0.8 mM). Transport reactions at 23 °C were started by the addition of [3H]melibiose (20 mCi/mmol) at a final concentration of 0.8 mM and terminated by a rapid dilution followed by immediate filtration. When tested, MTSEA+ (filled symbols) or MTSET+ (gray symbols) were added at a final concentration of 1 mM for 2 min before the addition of MelB substrates.

Chemical modification of single-Cys mutant with positively charged methanethiosulfonate derivatives (25) provides means to incorporate a positive charge at the Cys position in the mutants. Fig. 4 illustrates the effect of MTSEA+ or MTSET+ derivatives on the activity of R141C RSO membrane vesicles. Significant rescue of active transport (Fig. 4B), melibiose counterflow (Fig. 4A), or efflux (data not shown) was observed on reacting R141C vesicles with MTSEA+ (1 mM) but not with MTSET+ (1 mM). The selective reactivation of R141C permease by the permeant MTS reagent added externally to RSO membrane vesicles is readily explained by first taking into account that MTSEA+ traverses the E. coli membrane much more efficiently than MTSET+ at the concentration of 1 mM used here (30) and, second, that the thiol-reagent must cross the membrane to react with Arg-141 exposed to the cytoplasmic medium (7).

Analysis of R141K or R141Q permeases brought additional insights into the mechanism by which this charged arginine residue might be involved in MelB function. Fig. 5 and Table I indicate that R141Q behaved as R141C. Thus, R141Q failed to catalyze H+-, Na+-, or Li+-coupled transport of [3H]melibiose or [14C]TMG (Fig 5). However the mutant still bound alpha -[3H]NPG in a Na+-dependent fashion (Table I). Consistently, R141Q membranes displayed Na+-dependent FRET signal similar to that of R141C (data not shown) and no longer catalyzed carrier-mediated movements of sugar (counterflow, efflux, data not shown). The data in Table I and Fig. 5 also show that R141K permease retained ion-coupled melibiose or TMG transport and Na+-dependent NPG binding activity. However, it is worth noting that all Arg-141 substitutions led to limited reduction of sugar affinity (3-4-fold) in comparison to C-less MelB (Table I).


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Fig. 5.   alpha - or beta -galactoside transport by MelB permease carrying a substitution of a lysine or a glutamine for either Arg-141 or Arg-149. E. coli DW2-R cells expressing the R141K, R141Q, R149K, or R149Q mutants or the control C-less permease were prepared in Na+-free 0.1 M potassium phosphate (pH 7) containing less than 20 µM sodium salts as described under "Experimental Procedures." H+-coupled [3H]melibiose transport or [14C]TMG (3 mCi/mmol, 0.8 mM) were assayed in this Na+-free medium (50-µl aliquots, 100 µg of proteins) at 23 °C. Na+- or Li+-coupled transport of [3H]melibiose (20 mCi/mmol, 0.8 mM) were measured in the presence of 10 mM NaCl or LiCl. Histograms show the amount of labeled sugars accumulated in 10 min (means of 3 determinations ± S.E.).

Arg-149 Is Close or at the Sugar Binding Site-- All attempts to rescue R149C sugar binding or transport by reacting the mutant with either MTSEA+ or MTSET+ failed (see Fig. 6A). The strong aggregation tendency of purified R149C precluded any study of its accessibility to the SH reagents by currently available labeling procedures implying affinity purification of the permease. Analysis of R149K and R149Q were more instructive. Fig. 5 indeed shows that cells expressing the two last mutants catalyzed significant Na+-dependent [3H]melibiose accumulation. The positive charge of Arg-149 is, therefore, not an absolute requirement for active transport. However, some of the R149Q and R149K properties were significantly different from those of C-less MelB. Thus, although R149Q and R149K affinity for the coupling Na+ ions was not significantly modified (Fig. 5 and Table I), their affinity for alpha -galactosides was severely reduced (about 10-fold, Table I) and that for beta -galactosides was beyond detection limit. Overall, although a charged residue is not absolutely required at position 149 for active transport, the charged arginyl side chain seems to provide optimal interactions for co-substrate binding.


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Fig. 6.   Inhibition of Na+-dependent sugar transport of R149C/V343G in RSO membrane vesicles by MTSET+. RSO membrane vesicles (2 mg of protein/ml) harboring the revertant permease were equilibrated in Na+-free, 0.1 M potassium phosphate buffer (pH 7) and incubated for 5 min in the presence of MTSET+ (1 mM). [3H]Melibiose transport (20 mCi/mmol, 0.8 mM) was then assayed as described in Fig. 2 except for the presence of reduced phenazine methosulfate. Control experiments were carried out using C-less RSO membrane vesicles. Histograms represent the amount of labeled sugar accumulated in 10 min (means of 3 experiments ± S.E.).

Additional insight into the role of Arg-149 was obtained by examining the functional properties of second-site revertants of R149C. Twenty independent clones that recovered fermentation activity on MacConkey plates were isolated from four different experiments. Analysis of their nucleotide sequence indicated that 15 of them were true revertants. In contrast, the five remaining ones systematically carried a second substitution of a glycine for the valine 343 located in the inner half of helix X in addition to the original R149C mutation in loop 4-5 (see Fig. 1). Despite many attempts, no other distinct second-site suppressor mutation was isolated. Fig. 6 shows that R149C/V343G RSO membrane vesicles accumulated melibiose up to 10% of the levels observed in C-less MelB vesicles. Na+-dependent accumulation of [14C]TMG was about half that of [3H]melibiose in these vesicles (data not shown). Remarkably, although R149C/V343G had limited capacity to concentrate the substrates, it displayed sugar affinity and Na+ activation constants very close or identical to those of C-less MelB (Table I). Moreover, the revertant incubated with Dns2-S-Gal displayed a FRET signal comparable with the one recorded from C-less MelB (data not shown). One may finally note that the double mutant has a comparatively better affinity for alpha -galactosides (KD) or for Na+ ions on the basis of the KNa value than any of the individual single R149C or V343G mutants (Table I).

Finally, the effects of various thiol reagents on the transport and sugar binding properties of R149C/V343G RSO membrane vesicles were analyzed. First, we observed that reacting the RSO membrane vesicles with impermeant thiol reagents such as MTSET+ (Fig. 6) or PCMBS- (not shown) before the transport assay inhibited Na+-dependent melibiose transport by 75 and 50%, respectively. Permeant thiol reagents like MTSEA+ or N-ethylmaleimide inhibited the revertant transport activity by more than 90% (data not shown). Comparatively, sugar transport in control C-less membrane vesicles was at most reduced by less than 5% by MTSEA+ or not affected by the other reagents. To identify the step of the R149C/V343G transport cycle inhibited by MTSET+ inhibited, we analyzed the effect of this reagent on the alpha -[3H]NPG binding to RSO membrane vesicles and looked for an eventual protection by the co-substrates (Fig. 7). To this end, revertant RSO membrane vesicles were incubated in a medium without substrate, in media supplemented with either Na+ (20 mM) or alpha -[3H]NPG (4 µM) alone, or finally, in a medium supplemented with both substrates. Except for the control, MTSET+ was then added to all samples at a final concentration of 1 mM. After 5 min, the binding activity was assayed by the flow dialysis procedure in the presence of alpha -[3H]NPG and Na+ ions at a final concentration of 1 µM and 20 mM, respectively. The effect of the thiol reagent was estimated from the amount of bound ligand released in the medium upon the addition of melibiose in excess (20 mM). Fig. 7 shows that the amount of bound alpha -[3H]NPG released upon the addition of melibiose was comparable in membranes not incubated with MTSET+ (open circles) and in membranes treated with the thiol reagent in the presence of both Na+ and alpha -[3H]NPG (closed circles). Compared with this amount (100%), only 25% bound alpha -[3H]NPG was released when the membranes were incubated with MTSET+ in the absence of substrates (closed triangles) or when the medium contained only Na+ ions (open triangles). Finally, a significantly higher amount of alpha -[3H]NPG (50%, open diamonds) was released when the MTSET+ treatment was carried out in the presence of alpha -[3H]NPG alone. Comparatively, alpha -[3H]NPG binding to C-less membranes was insensitive to MTSET+ in all conditions tested (not shown). Taken together, these findings suggest that the sugar substrate affords protection against inactivation of sugar binding to the second site revertant by MTSET+. The more efficient alpha -[3H]NPG protection observed in Na+-containing and than in Na+-free media is most likely linked to the higher rate of sugar binding to MelB permease in the presence of Na+ ions (13). Finally, the lower extent of sugar binding inactivation in sugar-free medium was observed in RSO vesicles reacted with 0.2 mM PCMBS- (data not shown). Overall, these observations make it likely that the residue at position 149 is located at or close to the sugar binding site and in a hydrophilic environment accessible from the outer medium.


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Fig. 7.   Selective protection against inactivation by MTSET+ of alpha -[3H]NPG binding to R149C/V343G RSO membrane vesicles by the sugar. De-energized RSO membrane vesicles (20 mg of proteins/ml) were prepared in Na+-free medium (pH 7) containing 0.1 M potassium phosphate and separated in 5 aliquots (300 µl each). The following additions were made before MTSET+ treatment: closed circles, alpha -[3H]NPG (4 µM) and Na+ (20 mM); open diamonds, alpha -[3H]NPG (4 µM); open triangles, Na+ (20 mM); closed triangles, no substrates. Except for the control (open circles), all samples were incubated for 5 min with MTSET+ at a final concentration of 1 mM. The samples were then diluted four times, and when needed, the missing ion and/or sugar substrates were adjusted to a final concentration of 20 mM or 1 µM, respectively. After concentrating the protein to 20 mg/ml in the diluting medium, alpha -[3H]NPG binding was assayed by the flow dialysis procedure. When indicated by arrows, melibiose was added at a final concentration of 20 mM to the membrane suspensions. The dotted line refers to the rate of decay of free alpha -[3H]NPG recorded from membrane vesicles devoid of MelB permease.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The results presented above provide evidence that mutagenesis of two of four basic residues, Arg-141 and Arg-149, located in the inner loop 4-5 of MelB permease from E. coli impaired the Na+-sugar symporter function. The data suggest that Arg-141 primarily takes part in the reaction of co-substrate translocation, whereas the nearby Arg-149 residue may be located close or at the sugar binding site and most likely contributes to its structural organization. Together with the established properties of loop 4-5, these data bring evidence for a role of this extra-membranous cytoplasmic domain into the translocation process catalyzed by the ion-coupled sugar transporter.

The conclusion that only Arg-141 and Arg-149 are important for MelB function relies on the observation that individual replacement of either arginine with a cysteine totally inactivated MelB transport, whereas Cys substitution of the neighboring Lys-138 or Arg-139 residues had no noticeable effect. It is worth noting that Arg-141 and Arg-149 but not Lys-138 or Arg-139 are among the most conserved residues in the putative inner loop 4-5 of several homologous transporters composing the galactoside-pentoside-hexuronide family (6, 31, 32). Detailed analysis of the properties of Arg-141 and Arg-149 mutants suggest that these two residues contribute differently to MelB activity.

Although the substitution of a short (Cys) or larger (Gln) neutral residue for Arg-141 abolished sugar transport, it had no major effect either on the Na+-dependent recognition process of alpha - and beta -galactosides or on the associated cooperative modification of the sugar binding site environment. In contrast, R141C and R141Q no longer catalyzed energy-independent carrier-mediated flow of sugar in the inward (initial rate of counterflow) or in the outward (downhill efflux) direction. As emphasized under "Results," the influx of the labeled substrate during the initial phase of counterflow is part of an exchange process that only involves sugar loading/release at each membrane surface and shuttling of the loaded (MelB-ion-sugar) complex (3, 29). Because the R141C and R141Q mutants still bind the co-substrates at the outer surface, the mutant counterflow defect is most likely associated to a defective reorientation of the loaded carrier. Alternatively, the defect may arise from a reduction of the rate of release of the labeled sugar into the inner compartment (and/or a reduction of the rate of binding of internal sugars). It would be more difficult to link the lack of counterflow activity to an alteration of the binding properties of Na+ ions to MelB at the cytoplasmic membrane surface because the sugar exchange reaction occurs without concomitant binding and release of the coupling ion on the internal surface (33). Finally, the finding that only MelB carrying a positively charged residue (Arg, Lys, or R141C reacted with MTSEA+) at position 141 mediated co-substrate translocation highlights the importance of the charge. It raises the possibility of electrostatic interactions between Arg-141 and unknown negatively charged residues elsewhere in MelB. However, more complex hydrogen-bonding interactions like those observed in the arabinose-binding protein structure cannot be excluded (34). Screening of second-site suppressor revertants of R141C, so far unsuccessful, should help to discriminate among these interpretations.

Despite variable changes of MelB transport activity produced on replacing Arg-149 by a Cys, a Lys, or a Gln residue, all the mutations systematically modified MelB sugar recognition properties. Thus, the inactive R149C permease no longer bound sugars. Also, R149K and R149Q, which still catalyzed Na+-dependent active melibiose transport, displayed a selective and drastic reduction of their affinity for alpha -galactosides or no longer recognized beta -galactosides. Incidentally, because R149Q retained transport activity, contribution of the residue at position 149 to MelB activity is probably involved in hydrogen bond interactions rather than electrostatic ones. Implication of Arg-149 in the sugar binding reaction can, therefore, be suspected.

Analysis of the properties of the second-site suppressor mutant R149C/V343G provided further clues to the mechanism by which Arg-149 participates in the process of sugar binding. The presence of an additional mutation (V343G) led to partial recovery of transport activity despite the absence of the native arginine at position 149. This second mutation also restored an affinity for the substrates and a sugar binding site microenvironment comparable with that of the C-less permease. These findings provided independent confirmation that there is no absolute need for a charged residue at position 149 for sugar transport and recognition by MelB. Moreover, because the rescuing Val right-arrow Gly mutation occurred on a small apolar residue located on the hydrophobic face of helix X distant from the R149C mutation in loop 4-5, it seems more likely that the second mutation compensates for a structural defect prevailing in the R149C mutant. Taken together, the change in sugar recognition properties of the different Arg-149 mutants, the rescuing effect of a distant mutation in a permease still carrying the R149C mutation, and an unusual instability of R149C permease upon purification could be accounted for by assuming that Arg-149 is involved in the structural organization of the sugar binding site rather than in a direct binding interaction with the sugar.

The observed inhibition of both R149C/V343G sugar transport and binding activity by MTSET+ or PCMBS- provides insight into the relative localization of Arg-149 with respect to the sugar binding site. Thus, accessibility of the cysteine at position 149 to the two hydrophilic and impermeant thiol reagents in RSO membrane vesicles suggests that Cys-149 in the revertant and, by extension, Arg-149 in the C-less permease faces an intra-protein polar region communicating with the outer medium. Moreover, because the sugar itself selectively protected against inactivation of the sugar binding reaction by MTSET+ or PCMBS-, this polar region most likely corresponds to the sugar binding domain. Similar observations on several transporters have been already used as an indication that the specific region harboring the target residue is either directly involved in binding, lining the binding site, or at least conformationally coupled to the binding of substrates (20, 29, 35). In this context, it should be mentioned that a current topological model of MelB predicts Arg-149 localization at the junction of cytoplasmic loop 4-5 and helix V (Ref. 5; see Fig. 1). Any attempt to relocate Arg-149 within the adjacent membrane domain (helix V) is at best limited to the very inner extremity of this helix due to the strong helix-breaker motif PYP preceding Arg-149 in the MelB sequence (see Fig. 1). On this basis, one can predict that the sugar binding site region including Arg-149 lies down close or at the inner membrane surface. It is finally worth mentioning that Cys-scan mutagenesis of aligned residues in the polar sector of helices V and X suggests that the two helix inner-half portion lines the sugar binding site (manuscript in preparation).2

The two major conclusions drawn above are that Arg-141 is primarily involved in reorientation of the loaded carrier or in the process of sugar release at the inner membrane surface and that Arg-149 is a domain of the sugar binding site close to or at the inner membrane surface. Are these proposals consistent with known biochemical characteristics of Arg-141 and flanking regions of loop 4-5? It has previously been shown that Arg-141 is a melibiose-protected site of MelB labeling by a photoactivable azidophenyl sugar analog (24). It must be recalled that no residue in loop 4-5 and of the flanking helix V other than 141 was photolabeled, suggesting that Arg-141 is a specific labeling target in this MelB region. However, the reactive azido is at a short distance from the galactosyl moiety that dictates MelB sugar specificity (36). This means that the Arg-141 local region may well be adjacent to the sugar binding site rather than necessarily within the site. The observations that (i) Arg-141 is only 8 residues apart from Arg-149 in the primary amino acid sequence (Fig. 1), (ii) Arg-149 is located close to the inner water-membrane interface (Fig. 7), and (iii) Arg-141 is exposed to the inner aqueous medium (7) are in line with this interpretation. The two later findings further suggest that proximity between the Arg-141 domain of loop 4-5 and the Arg-149 domain of the sugar binding site occurs at the inner surface membrane level. Positioning Arg-141 in a domain immediately after the sugar binding site would also explain that replacing this arginine by neutral residues effects translocation or release of the sugar into the cytoplasm rather than binding of the external the sugar substrate to MelB. Finally, the finding that MelB substrates affords cooperative protection against loop 4-5 digestion by trypsin (Na+ and melibiose > Na+ melibiose) has been used to suggest that this loop undergoes structural rearrangement upon substrate binding (7). Loop 4-5 or part of it including the Arg-141 region may be regarded as a functional mobile domain, accessible to the bulk of the cytoplasm when the symporter is unloaded, and becomes inaccessible upon interaction with the nearby membrane sugar binding site once the carrier is loaded. Such a conformational transition of the Arg-141 region may favor either reorientation of the loaded carrier, transient interaction of Arg-141 with the sugar after reorientation of the permease, or finally, sugar release into the inner compartment. Consistently, previous fluorescence and mutagenesis studies of residues sitting in the N-terminal region of loop 4-5 and in the preceding helix IV suggest that this area of the transporter might behave as a hinge involved in the coupling mechanism of MelB (37). More work is, however, needed to assess the validity of these speculations. In conclusion, the data reported in this study provide ground evidence for the existence of a close relationship between loop 4-5 and the sugar binding site and strongly suggest that Arg-141 in the cytoplasmic loop 4-5 is catalytically involved in the process of Na+-coupled sugar translocation by the MelB symporter.

    ACKNOWLEDGEMENTS

We thank Raymonde Lemonnier for excellent technical assistance and Kerstin Meyer for comments on the manuscript.

    FOOTNOTES

* This work was supported in part by European Commission Grant Bio4-CT97-2119 and grants form the CNRS and the French Atomic Energy Commission.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger A post-doctoral fellow of the European Commission (Bio4-CT97-2119).

§ To whom correspondence should be addressed. Tel.: 33-4-93-76-52-12; Fax: 33-4-93-76-52-19; E-mail: leblanc@oceane.obs-vlfr.fr.

Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M210053200

2 C. Basquin, M. Abdel Dayem, R. Lemonnier, T. Pourcher, and G. Leblanc, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: MelB, melibiose permease, (6-O-alpha -D-galactopyranosyl-D-glucose); TMG, methyl-1-thio-beta -galactopyranoside; alpha -NPG, p-nitrophenyl alpha -D-6-galactopyranoside; FRET, fluorescence resonance energy transfer; Dns2-S-Gal, 2'-(N-dansyl)aminoethyl-1-thio-beta -D-galactopyranoside; MTSEA+, (2-aminoethyl)methanethiosulfonate hydrobromide; MTSET+, (2-(trimethylammonium)ethyl) methanethiosulfonate hydrobromide; PCMBS-, p-chloromercury benzoic acid; RSO membranes, right-side-out membranes.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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