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
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ABSTRACT |
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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.
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 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.
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 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
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- 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.
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.
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.
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).
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 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.
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 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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-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.
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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.
70 °C.
-maltoside
(DDM), with 0.5 mM phenylmethylsulfonyl fluoride and 15 mM
-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-
-D-glucopyranoside (Glycon GmbH), 30 mM imidazole, and 15 mM
-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-
-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.
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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.
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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.
Binding activity of substitutions with amino acids other than cysteine
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
( ) and mutant proteins T18S (
), A10C (
), and A10G (
) 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 (
) and mutant
proteins A10G (
), T18S (
), A10C (
), 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.
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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).
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.
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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
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.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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The abbreviations used are:
SMR, small multidrug
resistance;
TM, transmembrane domain;
DDM, n-dodecyl--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.
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