An Amino Acid Cluster around the Essential Glu-14 Is Part of the Substrate- and Proton-binding Domain of EmrE, a Multidrug Transporter from Escherichia coli*

Natalia Gutman, Sonia Steiner-Mordoch, and Shimon SchuldinerDagger

From the Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel

Received for publication, December 23, 2002, and in revised form, February 3, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

EmrE is a small multidrug transporter (110 amino acids long) from Escherichia coli that extrudes various drugs in exchange with protons, thereby rendering bacteria resistant to these compounds. Glu-14 is the only charged membrane-embedded residue in EmrE and is evolutionarily highly conserved. This residue has an unusually high pK and is an essential part of the binding domain, shared by substrates and protons. The occupancy of the binding domain is mutually exclusive, and, as such, this provides the molecular basis for the coupling between substrate and proton fluxes. Systematic cysteine-scanning mutagenesis of the residues in the transmembrane segment (TM1), where Glu-14 is located, reveals an amino acid cluster on the same face of TM1 as Glu-14 that is part of the substrate- and proton-binding domain. Substitutions at most of these positions yielded either inactive mutants or mutants with modified affinity to substrates. Substitutions at the Ala-10 position, one helix turn away from Glu-14, yielded mutants with modified affinity to protons and thereby impaired in the coupling of substrate and proton fluxes. Taken as a whole, the results strongly support the concept of a common binding site for substrate and protons and stress the importance of one face of TM1 in substrate recognition, binding, and H+-coupled transport.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Multidrug- and drug-specific efflux systems are responsible for clinically significant resistance to chemotherapeutic agents in pathogenic bacteria, fungi, parasites, and human cancer cells (2-4). Phylogenetic studies show that these efflux systems are associated with five superfamilies of transporters (5). One of these includes a family of small multidrug resistance (SMR)1-conferring proteins. The SMR family consists of small hydrophobic proteins of about 100 amino acid residues with four transmembrane alpha -helical spanners (6-8). These proteins function as oligomers (9, 10) and remove cationic drugs from the cytoplasm using a drug/H+ antiport mechanism (6-8). Genes coding for SMR proteins have been identified in many eubacteria and in some archaea (11, 12). The most extensively characterized SMR protein is EmrE, from Escherichia coli. The four transmembrane segments in EmrE are tightly packed in the membrane without any continuous aqueous domain, as was shown by cysteine scanning experiments (13). These results suggest the existence of a hydrophobic pathway through which the substrates are translocated. Glu-14, the only membrane-embedded charged residue is highly conserved in the SMR family (11). This residue has an unusually high pK and is an essential part of the binding domain, shared by substrates and protons (1, 14). The occupancy of the binding domain is mutually exclusive, and, as such, this provides the molecular basis for coupling of substrate and proton fluxes.

In TM1, where Glu-14 is located, a certain helical periodicity of sequence conservation is observed in the SMR family: (F/Y)L7XXA10(I/G)11XXE14(I/V)X(A/G/W)17(T/A/S/N)18XX(L/M) (11, 15), suggesting the functional importance of one face of the helix. Hsmr, a homologue from the archaeon Halobacterium salinarum, built from over 40% valine and alanine residues supplies further support for this contention. In Hsmr, the Ala and Val residues are clustered in domains that do not seem important for activity, and this dramatic phenomenon seems to reflect the outcome of an evolutionary process leading to the replacement of all residues not essential for function with either valine or alanine. In the TM1 of Hsmr, the face opposite Glu-14 is built almost entirely of Ala residues (12). We therefore experimentally probed the role of the residues in the same face of the helix as Glu-14 in substrate recognition and in stabilization of the negative charge in the membrane. We scanned these residues using site-directed mutagenesis and identified a cluster of five amino acids that play a role in substrate and H+ recognition and/or translocation. Substitutions at most positions yielded either inactive mutants or mutants with modified affinity to substrates. Substitutions at the Ala-10 position, one helix turn away from Glu-14, yielded mutants with wild type substrate binding properties but with modified affinity to protons. As a consequence, the mutants are impaired in the coupling of the proton gradient to substrate fluxes. The current study provides further insight into the coupling mechanism and stresses the importance of one face of TM1 in substrate recognition, binding, and H+-coupled transport.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial Strains and Plasmids-- E. coli JM109 (16) and TA15 (17) strains were used throughout this work. TA15 strain was previously transformed with plasmid pGP1-2, which codes for the T7 polymerase under the inducible control of the lambda  PL promoter (18). The plasmids used for EmrE gene expression are pT7-7 (18) derivatives with the histidine tag (EmrE-His) (1). The cysteine-less EmrE-His was built with serine (CLS-EmrE-His) or alanine (CAMY-EmrE-His) replacements.

Mutagenesis-- Mutants were obtained by polymerase chain reaction, using the overlap extension procedure as described by Ho et al. (19). For most of the mutations, a set of two overlapping oligonucleotide primers containing the desired mutation was constructed. The outside primers were those used for EmrE (20). The templates used were EmrE-His, CLS-EmrE-His, or CAMY-EmrE-His. In mutations at positions Tyr-6, Leu-7, Ala-10, Ile-11, Ala-13, and Glu-14, only two outside primers were used, and the forward one contained the desired mutation.

Mutagenic oligonucleotides were prepared incorporating a unique restriction site to facilitate mutant identification, and, in several cases, this required an additional conservative mutation. Mutated DNA was identified by the acquisition of the new restriction site. All of the PCR-amplified products were sequenced to ensure that no other mutations occurred during the amplification process.

Resistance to Toxic Compounds-- E. coli JM109 cells transformed with either pT7-7-EmrE-His, pT7-7 (vector), or with the various mutants were grown overnight at 37 °C in LB-Ampicillin medium. 5 µl of serial dilutions of the culture were spotted on a series of LB-Amp plates with various EmrE substrates (200 µg/ml ethidium bromide, 100 µg/ml acriflavine, and 0.1 mM methyl viologen) or on a plate with no addition. Growth was analyzed after overnight incubation at 37 °C.

Expression, Purification, and Reconstitution of EmrE-- E. coli TA15 cells transformed with plasmid pT7-7 were grown at 30 °C in minimal medium A supplemented with glycerol (0.5%), thiamine (2.5 µg/ml), ampicillin (100 µg/ml), and kanamycin (50 µg/ml). When the culture reached an A600 = 0.9, it was transferred to 42 °C for 15 min to induce the T7 polymerase. Then the culture was shifted back to 30 °C. 2 h later, the cells were harvested by centrifugation.

Cells were resuspended with buffer containing sucrose (250 mM), dithiothreitol (0.5 mM), NaCl (150 mM), Tris-Cl, pH 7.5 (10 mM), MgSO4 (2.5 mM), and DNase (15 µg/ml) (Sigma) and broken by a French press. The membrane fraction was collected by ultracentrifugation at 213,500 × g for 1 h at 4 °C and resuspended in the above buffer without dithiothreitol and without DNase. The membranes were frozen in liquid nitrogen and stored at -70 °C.

Reconstitution was performed essentially as described (21). 400 µl of membranes (containing 120 µg of EmrE-His protein) were solubilized in 2 ml of buffer containing 150 mM NaCl, 15 mM Tris-Cl, pH 7.5 (Na-buffer), 1.5% n-dodecyl-beta -maltoside (DDM), with 0.5 mM phenylmethylsulfonyl fluoride and 15 mM beta -mercaptoethanol. After removal of unsolubilized material by centrifugation (20,000 × g for 30 min), imidazole was added to 20 mM, and the EmrE-His protein was incubated with Ni2+-nitrilotriacetic acid beads (1 h, 4 °C). The beads were washed with at least 4 ml of Na-buffer, containing 1% n-octyl-beta -D-glucopyranoside (Glycon GmbH), 30 mM imidazole, and 15 mM beta -mercaptoethanol. The protein was eluted with 500 µl of the same buffer containing 200 mM imidazole and mixed with 375 µl of E. coli phospholipids mix (10 mg of phospholipids, 1.2% n-octyl-beta -D-glucopyranoside, 15 mM Tris-Cl, pH 7.5, and 150 mM NaCl). Eluted protein and phospholipids were sonicated together in a bath type sonicator until clear and diluted in buffer containing 0.19 M NH4Cl, 0.015 M Tris, pH 7.5, and 1 mM dithiothreitol. After 20 min at room temperature, samples were centrifuged at 250,000 × g for 60 min, and the pellet was resuspended in 100 µl of the same buffer, frozen in liquid nitrogen, and stored at -70 °C. Prior to the assay, the proteoliposomes were thawed at room temperature and sonicated lightly until clear.

TPP+ Binding Assay-- Tetraphenylphosphonium (TPP+) binding was assayed essentially as described (1). Ni2+-nitrilotriacetic acid beads (10 µl/assay) (Qiagen GmbH, Hilden, Germany) were washed twice in distilled water and once in 0.08% DDM/Na-buffer. Membranes, solubilized in 0.8% DDM/Na-buffer, were added to the washed beads and incubated at 4 °C for 1 h. The unbound material was discarded, and EmrE-His bound to beads was washed once with 0.08% DDM/Na-buffer. Buffer containing 5 nM [3H]TPP+ (27 Ci/mmol, Amersham Biosciences) was added, and the samples were incubated for 20 min at 4 °C with shaking. In each experiment, the values obtained in a control reaction, with 25 µM unlabeled TPP+, were subtracted. Separating the beads from the supernatant by pulse centrifugation stopped the binding reaction. The bead fraction was then incubated for 10 min at room temperature with 450 µl of 0.08% DDM/Na-buffer containing 150 mM imidazole in order to release the EmrE-His and [3H]TPP+ bound to it from the beads. After spinning down the beads, the [3H]TPP+-associated radioactivity was measured by liquid scintillation. All binding reactions were performed in duplicates. Each experiment was performed at least twice.

Transport Assay-- Uptake of [14C]methyl viologen into proteoliposomes was assayed at 25 °C by dilution of 3 µl of the ammonium chloride-containing proteoliposomes (100 ng of EmrE-His) into 200 µl of an ammonium-free solution (20). The latter contained 41 µM [14C]- methyl viologen (12.7-32.3 mCi/mmol; Sigma), 140 mM KCl, 10 mM Tricine, 5 mM MgCl2, and 10 mM Tris (pH 9.5). At given times, the reaction was stopped by dilution with 2 ml of the same ice-cold solution, filtering through Millipore GSWP filters (0.22 µm) and washing with an additional 2 ml of solution. The radioactivity on the filters was estimated by liquid scintillation. In each experiment, the values obtained in a control reaction, with 15 µM nigericin, were subtracted from all experimental points. The reaction was measured in triplicates. Each experiment was performed at least twice.

Efflux-- [14C]Methyl viologen was added (2 mM, 32.3 mCi/mmol; Sigma) into 60 µl of freshly thawed proteoliposomes (2 µg of EmrE-His) before sonication. After sonication, 3 µl of proteoliposomes (100 ng of the protein) were diluted into 200 µl of a solution containing 10 mM Tricine, 5 mM MgCl2, 190 mM NH4Cl, 15 µM nigericin, and 10 mM Tris (pH 8.5). At given times, the reaction was stopped by dilution with 2 ml of the same ice-cold solution, filtering through Millipore GSWP filters (0.22 µm), and washing with an additional 2 ml of solution. The radioactivity of the filters was estimated by liquid scintillation. In each experiment, the values obtained after 3 h were subtracted from all experimental points. The kinetics of efflux at 15 °C was measured in duplicates. Each experiment was performed at least twice.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In TM1, a relatively short 18-amino acid segment starting at Y4 and ending at M21, a periodical pattern of conservation is observed, with most of the conserved residues on the same face of the helix of the essential Glu-14 (11, 15). In Hsmr, an Smr protein from the archaeon H. salinarum, the face of TM1 opposite to Glu-14 is composed mostly of Ala residues (12). These findings suggested a central role of the residues in one face of TM1, and to experimentally probe it we performed a cysteine-scanning mutational analysis. The Cys-less-EmrE (CAMY-EmrE-His) protein displays significant transport activity and therefore provides a good starting point for generation of mutant proteins with single Cys insertions in replacement for residues in the proximity of Glu-14. Here we report the results obtained after replacing 12 amino acids in TM1. The activity of each of the mutant proteins has been tested both in vivo and in vitro.

A Large Fraction of the Residues in TM1 Cannot Be Replaced with Cys without Phenotype Impairment-- In vivo, the resistance conferred by each of the proteins was assessed by testing the ability of cells expressing them to grow under otherwise nonpermissive conditions. This was achieved in solid media containing either ethidium bromide (200 µg/ml), acriflavine (100 µg/ml), or methyl viologen (0.1 mM) in which 5 µl of logarithmic dilutions (103 to 105) of an overnight culture were spotted. Cells carrying the vector plasmid without any insert cannot grow in these media at any of the dilutions tested. Cells expressing either EmrE-His or CAMY-EmrE-His were able to grow at each of the dilutions. This assay provides a highly dynamic range to qualitatively analyze the activity of the mutants generated. The different strains clearly fall into two categories: those that grow in all three dilutions and those that do not. We arbitrarily assign a score of 1 to those strains capable of growing at dilutions higher than 103 (Fig. 1). Lack of growth at dilutions of 103 or less is defined as zero and is due to a nonfunctional transporter. This assay depends on the activity of EmrE, but no absolute quantitation can be made, because the levels of expression of EmrE do differ among the various mutants. Notwithstanding this limitation, several important conclusions can be made (Fig. 1). Six mutant proteins are incapable of conferring any measurable resistance: L7C, A10C, I11C, E14C, G17C, and T18C. They are located on the same face of the helix as Glu-14, and, in general, these are the locations of the conserved residues (Fig. 1). All of the other mutants tested are capable of conferring some degree of resistance to the various toxic compounds tested. The fact that such a large number of residues are nonpermissive is remarkable, since in several proteins, including other domains in EmrE, it has been previously observed that many residues can be replaced without loss of phenotypic complementation (13, 22-24). This finding, together with the conservation pattern observed, suggested that residues in the vicinity of Glu-14 are important for function, and we therefore studied the various mutants in detail.


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Fig. 1.   Mutations of the highly conserved residues in EmrE TM1 affect the growth phenotype. The ability of mutants at the indicated positions of TM1 to grow on solid media containing toxic compounds is shown in the upper histogram. The assay is described under "Experimental Procedures." A score of 1 is assigned to cells able to grow in the presence of toxic compounds at dilutions higher than 103; the lack of growth under these conditions is defined as zero; nd, not determined. The letters above the histograms represent the amino acid substitutions at each one of the positions. In the bottom image, the levels of conservation of the helix 1 residues are presented. Sequence alignment of TM1 from 63 EmrE homologs is represented as sequence logos (11). The scale gives the certainty of finding a particular amino acid at a given position and is determined by multiplying the frequency of that amino acid by the total information of that position. Orange, hydrophobic; gray, polar; green, amide; red, acidic; blue, basic. This figure is adapted from Ref. 11.

Expression and TPP+-binding Activity-- All of the mutant proteins were purified by Ni2+-nitrilotriacetic acid chromatography to assess expression levels and were then assayed for their ability to catalyze partial reactions of the transport cycle. Substrate binding was evaluated in the detergent-solubilized transporter using [3H]TPP+, a high affinity substrate (1). All of the mutants tested, including the ones incapable of conferring resistance, expressed to similar levels (not shown); however, the ability to bind [3H]TPP+ differed considerably among the various mutants. As expected, Y6C, L12C, A13C, V15C, I16C, and M21C, proteins that conferred resistance in the in vivo assay described above, were capable of binding [3H]TPP+ to levels at least 50% of that of the EmrE-His control or CAMY-EmrE-His (Fig. 2A). Whereas L7C, E14C, and G17C displayed no measurable binding activity (Fig. 2B) (1), other mutants bound [3H]TPP+ to various degrees (Fig. 2C), and their binding was further analyzed.


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Fig. 2.   One face of TM1 is important for TPP+ binding activity. The helical wheel projection of TM1 of EmrE is shown in the center. Positions of functional mutants in the phenotype assay are shown in gray; inactive mutants are shown in black. Positions that were not tested are shown in white. The results of [3H]TPP+ binding of the cysteine replacements are shown in histograms beside the TM1 wheel. The mutants are divided to three different types by their ability to bind TPP+: mutants functional in phenotype and in binding assays (A), inactive mutants (B), and mutants with negative phenotype but with partial binding activity (C). The [3H]TPP+ binding assay was performed as described under "Experimental Procedures." The results of bound [3H]TPP+ were calculated in pmol and normalized by EmrE concentration.

Mutants with Modified Affinity to TPP+-- Mutant proteins with substitutions at three positions, 10, 11, and 18, bound TPP+ (Fig. 2C), although they did not confer resistance (Fig. 1).

I11C bound 1.6 pmol of TPP+/µg of EmrE, about one-tenth of the TPP+ bound by the wild type under standard conditions (Fig. 2C). Further analysis of these results revealed that the reason for this decreased binding at 2.5 nM TPP+ is due to a decrease in affinity to 20 ± 2 nM, as compared with 1.9 nM for the wild type protein (10). To further probe this location, Ile-11 was replaced with Gly, a residue found in many of the other homologues (Fig. 1). However, I11G was even less active than I11C (Table I).


                              
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Table I
Binding activity of substitutions with amino acids other than cysteine
The experiment was performed as described under "Experimental Procedures" and was repeated twice in duplicates. The average and the average deviations are presented.

T18C showed low but consistent binding activity. Since it is partly conserved (Fig. 1), we constructed the T18S, T18A, and T18G mutations, amino acids present in this position in other homologues. These proteins did not confer resistance either (Fig. 1) and showed little binding activity except for T18S that bound about 1.1 pmol/µg EmrE (Table I). In this case too, the affinity to TPP+ was 21 ± 2 nM, 10 times lower than that displayed by the wild type protein.

Changes in the affinity to substrate measured in detergent-solubilized preparations affect also transport rates measured in reconstituted proteoliposomes. The T18S and I11C mutants, which display a lower affinity to TPP+, have moderate levels of uptake. Purified EmrE-His or mutant proteins were reconstituted into proteoliposomes, and the transport activity was measured. The Delta pH-driven [14C]methyl viologen uptake into proteoliposomes is shown in Fig. 3A. In this experiment, an artificial pH gradient is generated, and substrate transport against a concentration gradient is measured at a concentration of 41 µM (Km for the wild type protein = 260 µM) (11). Proteoliposomes reconstituted with EmrE-His rapidly accumulate substrate, and uptake levels off thereafter. The results for T18S are shown in Fig. 3A, where uptake after 5 min reaches a level of about 30% of EmrE-His. The results with I11C are not shown, but they are practically identical. This relatively lower accumulation capacity correlates well with the lower uptake rates and constant passive leaks. Lower transport rates are also detected in the downhill efflux assays (Fig. 3B). In these experiments, proteoliposomes are loaded with a high concentration of radioactively labeled substrate and diluted into an identical medium with no substrate. To prevent possible generation of proton gradients, the ionophore nigericin is added to the medium. The lower rates catalyzed by T18S (Fig. 3B) and I11C (not shown) are evident although these experiments are carried out at 2 mM methyl viologen, a relatively high concentration.


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Fig. 3.   Transport activity of mutants in TM1 reveals the importance of conserved residues in one face of the helix. A, uptake of [14C]methyl viologen into proteoliposomes reconstituted with EmrE-His or TM1 mutants. EmrE-His (black-square) and mutant proteins T18S (black-triangle), A10C (open circle ), and A10G (black-diamond ) were purified and reconstituted as described under "Experimental Procedures." Ammonium-loaded proteoliposomes (3 µl, containing 0.1 µg of EmrE) were diluted into an ammonium-free medium containing [14C]methyl viologen, pH 9.5, and radioactivity was incorporated at various times was measured. B, efflux activity of EmrE-His and the mutants of TM1. EmrE-His (black-square) and mutant proteins A10G (black-diamond ), T18S (black-triangle), A10C (open circle ), and E14C (*) were purified and reconstituted into proteoliposomes as described under "Experimental Procedures." Proteoliposomes were loaded with [14C]methyl viologen and diluted into substrate-free medium containing 15 µM nigericin to dissipate the proton gradient. The remaining substrate concentration inside the liposomes at various time points is shown as percentages of the initial value. The experiments were performed at pH 8.5, and one representative result of at least two independent repeats is shown.

Mutants with Modified Affinity to H+-- A strikingly different behavior was detected with mutants at position 10. A10C bound 5 pmol of TPP+/µg of EmrE, about 40% of the EmrE-His (Fig. 2C). A protein in which the replacement was with a smaller amino acid, A10G, bound 14 pmol of TPP+/µg of EmrE to the same levels as EmrE-His (Table I). Any other replacement at this position, A10S (an amino acid detected in this position in a few of the other homologues), A10L, A10V, and A10P resulted in inactive proteins.

The results described for Cys and Gly substitutions at position 10 suggested the possibility that these mutants bind substrate with high affinity but display a modified affinity to H+. The dependence of the binding and release of substrate on the ability of Glu-14 to release and bind protons is an essential element of the coupling mechanism (25, 26). TPP+ binding to wild-type EmrE requires deprotonation of Glu-14, whereas binding to E14D mutant is practically pH-independent in the range 6.5-8.5 (1). Shortening of the Glu side chain to Asp decreases the pKa of the carboxyl to around 5, compared with the unusually high pKa of about 7.5 in the EmrE-His (1). Thus, the E14D mutant catalyzes downhill efflux of substrate but cannot utilize the proton electrochemical gradient to drive uphill substrate accumulation (25, 26).

In this work, we tested whether mutants of TM1 modify the pKa of Glu-14 as judged from the pH dependence of TPP+ binding. [3H]TPP+ binding was measured at pH 6.5, and the values obtained for each mutant are shown in Fig. 4 as a percentage of the corresponding [3H]TPP+ binding at pH 8.6. The results demonstrate that most of the mutants bind less than 10% [3H]TPP+ at this pH, similar to what was previously shown for wild type EmrE (26). Strikingly, A10C and A10G display binding levels at pH 6.5 almost as high as at pH 8.6. These data indicate that the nature of the amino acid at position 10 influences the pK of Glu-14 and suggest a close interaction with Glu-14. Since, as mentioned above, the pH dependence of binding reflects the coupling between proton and substrate fluxes, transport functions of A10C and A10G were investigated.


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Fig. 4.   Relative [3H]TPP+ binding at pH 6.5 in mutants of residues in TM1. The [3H]TPP+ binding procedures were done as described under "Experimental Procedures," with the exception that a fraction of the immobilized protein was washed twice with buffer containing 150 mM NaCl, 0.08% DDM, and 30 mM Mes-Tris, pH either 6.5 or 8.6, and the labeled TPP+ was added in the presence of the same buffer of appropriate pH. The relative level of [3H]TPP+ binding at the low pH (pH 6.5) is shown as percentage of the control [3H]TPP+ binding at high pH (pH 8.6).

The "Uncoupled" Mutants A10C and A10G Cannot Utilize pH Gradients to Drive Substrate Transport Uphill-- Similar to what was observed with E14D, the two mutants with pH-independent binding, A10C and A10G, have no uptake activity. The Delta pH-driven [14C]methyl viologen uptake into proteoliposomes was assayed as described above and shown in Fig. 3A. Whereas proteoliposomes reconstituted with EmrE-His rapidly accumulate substrate, those with either A10C or A10G showed no accumulation whatsoever. The same proteoliposomes were used to assay the ability of the various mutants to catalyze H+-independent downhill efflux. A10C displays a high rate of downhill efflux of [14C]methyl viologen (40 pmol/s/µg of EmrE), comparable with that detected for EmrE-His (Fig. 3B). This behavior is very similar to that reported for E14D, a mutant that catalyzes downhill efflux but is incapable of utilizing the proton electrochemical gradient (25). In addition, both E14D and A10C have a more moderate pH dependence of the efflux rate than that of the wild type (not shown). Strikingly, A10G has a much lower efflux rate than A10C, although it binds TPP+ with similar affinity. The efflux rate is 6.4 pmol/s/µg of EmrE, about 10% of that observed with the wild type protein. To test whether the return of the unloaded transporter is impaired, exchange was tested by the addition of saturating concentrations of unlabeled methyl viologen to the medium as in Ref. 25. The exchange rate was only slightly higher than the efflux rate (data not shown). Thus, whereas mutants A10C and E14D behave very similarly, the A10G mutant displays a novel behavior. All three bind TPP+ in a pH-independent mode and are, therefore, affected in the proton coupling mechanism. However, A10C and E14D efflux the substrate down its electrochemical gradient, but A10G has very slow downhill efflux activity. This suggests that it cannot undergo the conformational change(s) necessary for substrate transport following binding.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Systematic cysteine-scanning mutagenesis of the neighbors of residue Glu-14 in TM1 revealed an amino acid cluster surrounding Glu-14 that is part of the substrate-binding domain. Several types of mutants were obtained (Fig. 5): 1) wild type phenotype, wild type substrate binding (positions shown in gray: Tyr-6, Leu-12, Ala-13, Val-15, Ile-16, and Met-21); 2) negative phenotype, no substrate binding (Leu-7 and Gly-17); 3) negative phenotype, substrate binding with lower affinity (Thr-18 and Ile-11); and 4) negative phenotype, wild type substrate binding, modified affinity to H+, uncoupled transport (A10).


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Fig. 5.   Summary of the phenotype of substitutions in TM1. The helical wheel projection of the first transmembrane domain is shown. Positions that are sensitive to mutations are shown in black; positions that are insensitive to the mutations are shown in gray; positions that were not mutated are in white.

The residues that yielded proteins with wild type phenotype and bound the substrate TPP+ to wild type levels were located on the face of the helix opposite of Glu-14 (Leu-12, Ala-13, Val-15, and Ile-16) or far enough from Glu-14 on the same face (Tyr-6 and Met-21). We conclude that these domains are not involved in either one of the functions required for catalysis of transport. These results provide experimental support for the prediction put forward based on the lack of conservation of residues in these positions (11). A remarkable and very graphic demonstration of the evolutionary argument was provided with the analysis of the sequence of Hsmr, the archaeal homologue of EmrE. Hsmr displays a remarkable amino acid composition of over 40% valine and alanine residues. The distribution of valine and alanine residues within the transmembrane domains of Hsmr is not random. Many of these abundant residues appear to be clustered in structural domains that are not essential for activity. This resembles the result of an alanine-scanning mutagenesis experiment, pointing out instantaneously the residues that are important for the function of the protein and therefore cannot be replaced with valine or alanine. In TM1 of Hsmr, all residues in the face opposite that of Glu-14 are either alanine or valine (12).

Only two of the cysteine substitutions in TM1 that were phenotypically inactive were also inactive in the binding assay. Those were the mutations of the highly conserved Leu-7 or the less conserved Gly-17.

Mutations at either one of two positions 11 and 18 decreased the affinity to substrate about 10-fold (Fig. 5). At the relatively conserved position 11, Gly is found in quite a number of homologues. However, the I11G mutation was completely inactive. On the other hand, cysteine replacement at this position generates a protein with a modified affinity. The I11C protein can also utilize the pH gradient to transport methyl viologen against its concentration gradient. At position 18, replacement of Thr with Cys or Ala, another residue found at this position in other homologues, yielded an inactive protein. Replacement with Ser, another hydroxyamino acid, generated a protein with a decreased affinity to TPP+ but capable of accumulating methyl viologen in a process driven by a pH gradient. Interestingly, although capable of energy-driven transport, neither I11C nor T18S can confer resistance to the tested drugs. We reason that because of their decreased affinities, neither one can lower the concentration of the toxic compounds below the cytotoxic levels.

Most revealing were the replacements at position 10 (Fig. 5). The Cys substitution yielded a protein with a modified affinity to H+ as judged from the pH independence of TPP+ binding in the range 6.5-8.6. We previously showed that the pH dependence of TPP+ binding is a reflection of the pK of the carboxyl at position 14 (1, 21). Thus, TPP+ binds to EmrE after H+ release from Glu-14, and it is released from the binding site upon protonation of Glu-14. This property provides the basis of coupling between H+ and substrate movements (14, 26). A protein with a pK below the "physiological" range cannot utilize pH gradients for substrate accumulation, but it catalyzes downhill transport at rates similar to that of wild type. This was previously shown for an E14D mutation and is now shown for substitutions at position 10. To analyze the mechanism by which this occurs, several other replacements were engineered. A10S, isosteric to Cys, yielded an inactive protein; hydrophobic replacements A10V and A10L were ineffective, as was also A10P, a smaller residue that may introduce a kink and confers some rigidity to the helix. A very interesting behavior was detected in the A10G mutant. Similarly to A10C, TPP+ binding to A10G displays high affinity but is pH-independent. As a consequence, the protein does not catalyze Delta pH-driven accumulation. Surprisingly, however, A10G is impaired also in efflux and exchange rates. The exchange reaction, unlike downhill transport, involves only translocation of the substrate-transporter complex (see Ref. 25 for kinetic scheme). Since the affinity of A10G to TPP+ was practically identical to that of the wild type as were the rates of TPP+ release from the protein (measured as in Ref. 1; not shown), the results, taken as a whole, suggest that A10G is impaired in the translocation step of the substrate-transporter complex.

The environment around Glu-14 is chemically unique as judged by its unusually elevated pKa. Recent advances in the structural analysis of multidrug recognizing transcription factors suggest that these proteins possess large hydrophobic binding sites and bind their substrates through a combination of hydrophobic and electrostatic interactions (27, 28). Negative charges in these hydrophobic domains are stabilized by hydrogen bonding to other residues such as the hydroxyls in tyrosines (29). In EmrE, we have suggested that the negative charge of Glu-14 may be stabilized in part by interaction with the corresponding residue of a neighboring monomer (14). The effects of Ala-10 substitutions on the pK of Glu-14 may be due to changes in the nature of this interaction; Cys is bulkier than Ala and may push apart the two helices. The smaller Gly residues may allow greater flexibility and may thus decrease the time that Glu-14 residues in two neighbor monomers interact. Any other replacements at position 10 yielded nonfunctional proteins, suggesting again a central role for Ala-10. A close interaction between a carbonyl moiety with a high pKa and a neighbor Ala has been suggested in the case of subunit c of the H+/ATPase. In this protein, it has been suggested that two Ala residues together with an Ile and an Asp interact to form the H+ binding site in the subunit c-oligomer (30).

The results described in this work strongly support the predictions from the analysis of evolutionary conservation and the amino acid sequence of Hsmr. They provide compelling evidence for the involvement of a cluster of amino acids around Glu-14 in substrate and H+ recognition in EmrE.

    FOOTNOTES

* This work was supported by grants from the Deutsche-Israeli Program (Federal Ministry of Education, Science, and Research, International Bureau at the German Aerospace Center Technology), National Institutes of Health Grant NS16708, and Israel Science Foundation Grant 463/00.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 To whom correspondence should be addressed. Tel.: 972-2-6585992; Fax: 972-2-5634625; E-mail: Shimon.Schuldiner@huji.ac.il.

Published, JBC Papers in Press, February 16, 2003, DOI 10.1074/jbc.M213120200

    ABBREVIATIONS

The abbreviations used are: SMR, small multidrug resistance; TM, transmembrane domain; DDM, n-dodecyl-beta -maltoside; TPP+, tetraphenylphosphonium; EmrE-His, EmrE tagged with Myc epitope and six His residues according to Ref. 1; CLS-EmrE-His, EmrE in which the three native cysteine residues were replaced with Ser; CAMY-EmrE-His, EmrE in which the three native cysteine residues were replaced with Ala; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Mes, 4-morpholineethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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