A Promiscuous Conformational Switch in the Secondary Multidrug Transporter MdfA

Abstract

Multidrug (Mdr) transporters are membrane proteins that actively export structurally dissimilar drugs from the cell, thereby
rendering the cell resistant to toxic compounds. Similar to substrate-specific transporters, Mdr transporters also undergo
substrate-induced conformational changes. However, the mechanism by which a variety of dissimilar substrates are able to induce
similar transport-compatible conformational responses in a single transporter remains unclear. To address this major aspect
of Mdr transport, we studied the conformational behavior of the Escherichia coli Mdr transporter MdfA. Our results show that indeed, different substrates induce similar conformational changes in the transporter.
Intriguingly, in addition, we observed that compounds other than substrates are able to confer similar conformational changes
when covalently attached at the putative Mdr recognition pocket of MdfA. Taken together, the results suggest that the Mdr-binding
pocket of MdfA is conformationally sensitive. We speculate that the same conformational switch that usually drives active
transport is triggered promiscuously by merely occupying the Mdr-binding site.

Full text

A Promiscuous Conformational Switch in the Secondary
Multidrug Transporter MdfA
*
Received for publication, July 30, 2009, and in revised form, September 22, 2009 Published, JBC Papers in Press, October 5, 2009, DOI 10.1074/jbc.M109.050658
Nir Fluman, Devora Cohen-Karni, Tali Weiss, and Eitan Bibi
1
From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Multidrug (Mdr) transporters are membrane proteins that
actively export structurally dissimilar drugs from the cell,
thereby rendering the cell resistant to toxic compounds. Similar
to substrate-specific transporters, Mdr transporters also
undergo substrate-induced conformational changes. However,
the mechanism by which a variety of dissimilar substrates are
able to induce similar transport-compatible conformational
responses in a single transporter remains unclear. To address
this major aspect of Mdr transport, we studied the conforma-
tional behavior of the Escherichia coli Mdr transporter MdfA.
Our results show that indeed, different substrates induce similar
conformational changes in the transporter. Intriguingly, in
addition, we observed that compounds other than substrates
are able to confer similar conformational changes when
covalently attached at the putative Mdr recognition pocket of
MdfA. Taken together, the results suggest that the Mdr-bind-
ing pocket of MdfA is conformationally sensitive. We specu-
late that the same conformational switch that usually drives
active transport is triggered promiscuously by merely occu-
pying the Mdr-binding site.
Mdr
2
transporters are membrane proteins that expel a wide
spectrum of chemically dissimilar drugs from the cell, thereby
rendering it resistant to multiple drugs. They exist in all king-
doms of life and constitute a major mechanism underlying
bacterial resistance to antibiotics and cancer resistance to
chemotherapy. In addition to their clinical importance, Mdr
transporters pose intriguing biochemical questions because of
their multispecificity and their capacity for catalyzing coupled
transport reactions with an extraordinarily broad spectrum of
substrates.
Mdr transporters exist in many families of transport proteins
that utilize various transport mechanisms (1, 2). Interestingly,
however, Mdr transporters from different families share many
similar mechanistic features (3, 4), suggesting that certain
aspects of multispecific transport by transporters having differ-
ent structures and families are similar. The research of recent
years has shed light on the major biochemical properties of Mdr
transporters. These transporters contain large substrate-bind-
ing pockets and can extract their substrates from the cytoplasm
and/or the membrane. Additionally, it appears that hydropho-
bic and electrostatic interactions underlie multispecific drug
binding (reviewed in Ref. 3). Nevertheless, the mechanism
underlying transport and coupling remains to be elucidated.
Transporters function by alternating between conforma-
tional states; for efficient coupling the transporter must be able
to conformationally respond to substrate binding. In the case of
Mdr transporters, this situation is intriguing because they
should be able to produce one or more transport-competent
conformational responses that fit a variety of chemically and
structurally unrelated substrates. Do all substrates induce the
same conformational change? Or do dissimilar substrates
induce different conformational changes, all of which facilitate
transport? Although it was shown that substrate binding indeed
induces conformational changes in Mdr transporters (5, 6), it is
not understood how a single transporter can be “conformation-
ally responsive” to the binding of a diverse group of compounds,
such that all of them induce structural rearrangements in the
protein that facilitate transport.
Here we studied this question by utilizing several approaches
for detecting conformational changes in the Escherichia coli
Mdr transporter MdfA, which serves as a model of secondary
Mdr transport (7). MdfA is a member of the major facilitator
superfamily, which constitutes the largest family of transport-
ers (8) and is the most prominent family of bacterial drug trans-
porters, many of which function as drug/H⫹antiporters (3).
The study reported here suggests that dissimilar substrates
indeed induce similar conformational changes in MdfA. Addi-
tionally, we show that even nonsubstrate compounds can
induce related conformational changes, provided that they are
forced to bind at the putative Mdr recognition pocket of MdfA.
Thus, the conformational changes are generated promiscu-
ously. We conclude that MdfA has a sensitive conformational
switch that can be triggered either by substrate binding or by
attaching unrelated agents inside the pocket.
MATERIALS AND METHODS
Plasmids—Plasmids for overexpression of MdfA or various
single cysteine mutants have been previously described (6, 9).
pT7–5/Para/mdfA
6HIS
encoding the single cysteine mutant
G410C was generated utilizing a standard PCR method with
mutagenic oligonucleotide primers and a plasmid encoding
Cys-less MdfA as a template. The plasmid was sequenced to
verify that only the desired mutation was inserted.
*This work was supported in part by the Israel Cancer Research Foundation,
the Minerva Foundation, and the Israel Science Foundation.
1
To whom correspondence should be addressed. Fax: 972-8-9346334; E-mail:
e.bibi@weizmann.ac.il.
2
The abbreviations used are: Mdr, multidrug; PMSF, phenylmethanesulfonyl flu-
oride; DDM,
␤
-dodecyl maltopyranoside; Mal-PEG, maleimide-polyethyleneg-
lycol; Cm, chloramphenicol; PK, proteinase K; mBBr, monobromobimane;
NEM, N-ethylmaleimide; AMS, 4-acetamido-4⬘-maleimidylstilbene-2,2⬘-disul-
fonic acid; IAA, iodoaceteadime; MTSEA, 2-aminoethyl methanethiosulfonate;
MTSES, 2-sulfonatoethyl methanethiosulfonate; MTSMT, (trimethylammoni-
um)methyl methanethiosulfonate; TPP, tetraphenylphosphonium.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 47, pp. 32296 –32304, November 20, 2009
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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Overexpression of MdfA and Preparation of Membranes—
E. coli cells harboring plasmid pUC18/Para/mdfA
6HIS
or pT7–
5/Para/mdfA
6HIS
were grown at 37 °C in LB medium supple-
mented with ampicillin (200
␮
g/ml). Overnight cultures were
diluted to 0.07 A
600
units and grown to 1.0 A
600
units, and the
culture was then induced with 0.2% arabinose for 1 h. A typical
10-liter culture yielded ⬃15 g (wet weight) of cells. Cell pellets
were washed once in 150 ml of 50 mMpotassium P
i
(pH 7.3)
supplemented with 2 mMMgSO
4
, and collected by centrifuga-
tion (15 min, 5,000 ⫻g). Next, the cells were suspended in 90 ml
of the same buffer containing 10
␮
g/ml DNase and 1 mMPMSF
and passed three times through a liquidizer (Emulsiflex-C5;
Avestin) (10,000 p.s.i.) for disruption. Cell debris was removed
by centrifugation (30 min, 8,000 ⫻g), and the membranes
were collected by ultracentrifugation (1 h, 100,000 ⫻g). The
membranes were homogenized in 27 ml of urea buffer (20
mMTris-HCl, pH 8, 0.5 MNaCl, 5 Murea, 10% glycerol, 28
mM
␤
-mercaptoethanol, and 1 mMPMSF), incubated for
30 min at 4 °C, and collected by ultracentrifugation (2.5 h,
100,000 ⫻g). The membranes were washed with 27 ml of
membrane buffer (20 mMTris-HCl, pH 8, 0.5 MNaCl, 10%
glycerol, and 3.5 mM
␤
-mercaptoethanol). Finally, the mem-
branes were suspended by homogenization in 7 ml of the
same buffer, and aliquots of 1 ml were snap-frozen in liquid
nitrogen and stored at ⫺80 °C.
Modification of Single Cysteine Mutants by Sulfhydryl
Reagents—The membranes were thawed quickly at 37 °C and
transferred to ice, collected by ultracentrifugation (1 h,
100,000 ⫻g), resuspended, and homogenized in an equal vol-
ume of reaction buffer (20 mMTris-HCl, pH 7, 0.5 MNaCl, 10%
glycerol). Sulfhydryl reagents (final concentration, 2 mM) were
added, and the suspension was sonicated (twice for 10 s using a
probe sonicator) to allow distribution of the reagent on both
sides of the membrane. The reaction was completed overnight
by tilting at 4 °C. When light-sensitive reagents were used, the
reaction and subsequent steps of the experiments were done in
the dark to minimize bleaching.
Membrane Solubilization and MdfA Purification—The
membranes were diluted to 2.7 ml in solubilization buffer
(20 mMTris-HCl, pH 8, 0.5 MNaCl, 10% glycerol, 0.1%
␤
-dode-
cyl maltopyranoside (DDM), 5 mMimidazole) and solubilized
by the addition of 0.33 ml of 10% DDM (final concentration,
1.1%). Insoluble material was discarded by ultracentrifuga-
tion (30 min, 100,000 ⫻g), and the soluble fraction was
mixed with solubilization buffer-equilibrated Talon beads
(Clontech) (0.25 ml). Next, the mixture was agitated for 3 h
at 4 °C, and the suspension was poured into a column. The
column was then washed (2 ⫻2 ml of solubilization buffer).
MdfA was eluted in 0.75 ml of elution buffer (20 mMTris-
HCl, pH 7.2, 0.12 MNaCl, 10% glycerol, 0.1% DDM, 100 mM
imidazole) after initial discard of 0.125 ml. The protein was
then dialyzed overnight against dialysis buffer (20 mMTris-
HCl, pH 7.2, 0.12 MNaCl, 10% glycerol, 0.01% DDM) at 4 °C.
The protein concentration was determined spectrophoto-
metrically by measuring A
280
.
Assessing the Degree of Chemical Modification Utilizing
Maleimide-Polyethyleneglycol (Mal-PEG)—The proteins were
denatured by 1% SDS to assist the exposure of cysteine residues.
To test the degree of free cysteines, the protein was allowed to
react with Mal-PEG (5000 kDa) for 2–3 h at room temperature.
The products were analyzed by SDS-PAGE (12.5%), followed by
Coomassie staining. The Mal-PEG-MdfA adduct appeared
⬃20 kDa heavier than MdfA or MdfA modified by any of the
test sulfhydryl reagents.
Binding of [
3
H]TPP—The binding assays were performed
essentially as described (9) with the following modifications.
Purified protein (typically 30
␮
g) was mixed with nickel-nitrilo-
triacetic acid beads (150
␮
l) in 5 ml of buffer A (20 mMTris-
HCl, pH 8, 0.5 MNaCl, 10% glycerol, 5 mMimidazole, 0.1%
DDM) and gently agitated for 30 min at 4 °C. The unbound
material (supernatant) was discarded by brief centrifugation (2
min, 700 ⫻g). The beads were resuspended in 1.5 ml of buffer C
(20 mMTris-HCl, pH 7, 0.5 MNaCl, 0.1% DDM), divided into
100-
␮
l aliquots, and mixed with 100
␮
l of substrate containing
solutions (in buffer C) to yield a final concentration of 10 nM
[
3
H]TPP (2 Ci/mmol) either in the presence or absence of 1 mM
cold TPP or 0.5 mMchloramphenicol (Cm). The mixture was
incubated by tilting for 10 min at 4 °C. 180
␮
l of the resin was
then transferred to a Promega Wizard minicolumn on top of a
microcentrifuge tube and centrifuged at 10,000 ⫻gfor 20 s.
Unbound (flow-through) material was discarded, and the resin
was resuspended in 100
␮
l of buffer C containing 350 mMimid-
azole. The radioactivity of this suspension was measured by
liquid scintillation. The amount of [
3
H]TPP bound to the resin
in the absence of MdfA was subtracted from all measurements.
The amount of bead-bound protein was determined for each
of the differently modified proteins for comparison, by ana-
lyzing the eluates by SDS-PAGE, Coomassie staining, and
densitometry.
Fluorescence Measurements—For spectra measurements,
MdfA was diluted to 1
␮
Min binding buffer (20 mMTris-HCl,
pH 7.2, 0.12 MNaCl, 10% glycerol, 0.1% DDM), transferred to a
quartz cuvette, and preheated to 30 °C in the fluorimeter (Var-
ian Cary Eclipse fluorescence spectrophotometer). The emis-
sion spectra were recorded using excitation wavelengths of 280
or 375 nm for tryptophan or mBBr fluorescence, respectively, in
the presence or absence of 50
␮
MTPP. The inner filter effect
introduced by the slight absorption of TPP was corrected as
described (10).
For TPP binding experiments, MdfA was diluted in binding
buffer to 0.1–0.25
␮
M. A prewarmed MdfA sample (2 ml at
30 °C) was continuously stirred in a quartz cuvette throughout
the experiment. The fluorescence was measured continuously,
and the sample was titrated by the addition of 10-
␮
l TPP ali-
quots (in binding buffer) to yield the indicated final concentra-
tions and incubated with TPP for 1 min, and the fluorescence
was measured. Excitation/emission wavelengths for trypto-
phan and mBBr fluorescence were 280/340 and 375/460 nm,
respectively. The TPP-induced inner filter effect was corrected
in the tryptophan fluorescence measurements as described
(10). The concentration of Cm was kept tightly constant to
avoid correcting for Cm light absorption. The measurements
were done in triplicate, and fluorescence of buffer/TPP solu-
tions containing no MdfA was subtracted. The results were
fitted to the following binding function using the nonlinear
regression software LABfit
TM
,
Conformational Switch in MdfA
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%⌬F⫽%⌬Fmax ⫻关TPP兴
Kd⫹关TPP兴(Eq. 1)
where %⌬Fis the percentage of fluorescence change (quench-
ing or increase), %⌬F
max
represents %⌬Fin a saturating TPP
concentration, and K
d
is the dissociation constant.
Limited Proteolysis of MdfA by Proteinase K (PK)—20
␮
lof
MdfA (0.15
␮
g/ml in binding buffer) were mixed with 3
␮
lof
the substrate to yield 50
␮
MTPP or 1 mMin the case of other
substrates/compounds. To initiate proteolysis, 3
␮
l of 5 mg/ml
PK (in 20 mMTris-HCl, pH 8, 2 mMCaCl
2
) were added and
mixed. Proteolysis was typically allowed to proceed for 10 min
or several hours at 4 °C. The reaction was quenched by the
addition of 2
␮
l of 200 mMPMSF followed by incubation at
room temperature (10 min). The cleaved protein was analyzed
by 12.5% SDS-PAGE and Coomassie staining.
RESULTS
Site-specific Labeling of MdfA by mBBr Stimulates Substrate
Binding—We have previously employed a genetic screen to
study how MdfA interacts with its substrates (6). This assay
identified residues that potentially participate in Mdr recogni-
tion. When viewed on a three-dimensional model of the trans-
porter (11), most of the genetically identified residues appear to
line a central pathway in MdfA, proposed to constitute an Mdr
recognition pocket (Fig. 1). Several of the genetically identified
sites have been studied in more detail, and residue Ala
147
was
found important for interaction of the transporter with Cm (6).
To investigate substrate binding further, we labeled the puta-
tive pocket by a fluorescence probe covalently bound at posi-
tion 147, utilizing a functional Cys-less mutant with a single
cysteine inserted at this position (MdfA-A147C). To this end,
we used the cysteine-reactive fluorescent compound mBBr
(12), which also proved useful for studying integral membrane
proteins (13). Labeling was accomplished by incubating iso-
lated membranes with mBBr, and the mBBr-MdfA adduct was
purified (Fig. 2A). The efficiency of mBBr labeling was analyzed
using a second sulfhydryl reagent, Mal-PEG, which increases
the mass of the protein upon covalent binding. When mixed
with the unlabeled MdfA-A147C mutant, Mal-PEG reacted
with the protein and generated a single, higher molecular
weight adduct, consistent with the presence of one reactive cys-
teine in the protein (Fig. 2A,lane 2). In contrast, when MdfA-
A147C was prelabeled by mBBr, the reaction with Mal-PEG
was blocked (Fig. 2A,lane 4), suggesting that the cysteine at
position 147 was already occupied by mBBr. Thus, a large frac-
tion of MdfA-A147C appears to be labeled by mBBr. To deter-
mine whether the modified protein is functional, we compared
the substrate binding capabilities of the purified labeled and
unlabeled MdfA-A147C. Surprisingly, we observed that the
mBBr-labeled transporter binds more TPP (an MdfA substrate)
than the unlabeled protein (Fig. 2B,gray bars). Because MdfA in
our experiments is nearly pure (Fig. 2A), this indicates that the
modification by mBBr stimulated TPP binding by MdfA.
Previous work on MdfA showed that it can interact with two
substrates, Cm and TPP, simultaneously and that binding of
Cm stimulates TPP binding to MdfA (9). To test the potential
relevance to our results with the mBBr-labeled protein, we
measured the effect of Cm on TPP binding by the purified,
labeled, and unlabeled MdfA-A147C proteins. Indeed, as
observed previously with wild-type MdfA, Cm also stimulated
the binding of TPP to MdfA-A147C but to a lesser extent, pos-
sibly because cysteine in this position slightly impairs the inter-
action with chloramphenicol (6). In contrast, however, Cm had
merely a minor effect on TPP binding to the mBBr-labeled pro-
tein (Fig. 2B). This slight effect might reflect stimulation of a
small fraction of the protein that was not labeled by mBBr. The
diminished effect of Cm on TPP binding indicates that the
stimulatory potential of Cm had been exploited by mBBr label-
ing at this site. In addition, it is also likely that Cm binding was
blocked by the covalently bound mBBr. Notably, the effects of
mBBr and Cm were quantitatively similar (Fig. 2B), suggesting
FIGURE 1. Putative substrate binding residues viewed on the three-di-
mensional model of MdfA. The ribbon representation of MdfA (11) shows
Glu
26
, and those residues that were studied here at position 147, 335, and 410
(black spheres). The residues shown as gray spheres are putatively involved in
substrate recognition by MdfA (6).
FIGURE 2. Purification and TPP binding activity of mBBr-labeled MdfA-
A147C. A, membranes from cells overexpressing MdfA-A147C were incu-
bated in the presence or absence of mBBr. MdfA was purified to homogeneity
and treated as described under “Materials and Methods” with (lanes 2 and 4)
or without (lanes 1 and 3) Mal-PEG, separated by SDS-PAGE, and stained by
Coomassie. Mal-PEG adduct is indicated by an arrow.B,[
3
H]TPP binding by
mBBr labeled or unlabeled MdfA-A147C with or without a saturating concen-
tration of Cm or excess of cold TPP.
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that mBBr labeling and Cm binding affect the transporter sim-
ilarly (see below).
mBBr Mimics the Effect of Cm on MdfA Only When
Covalently Bound at the Putative Mdr Recognition Pocket—As
demonstrated previously, the stimulatory effect of Cm on TPP
binding is due to an increased affinity of the binary complex
MdfA-Cm for TPP (9). To further characterize and compare
the effects of Cm and mBBr on MdfA-A147C, we measured the
affinity for TPP by utilizing two fluorimetric assays. With the
unlabeled MdfA-A147C, we used intrinsic fluorescence mea-
surements, based on a reduction (of ⬃10%) in fluorescence
upon TPP binding (Fig. 3A). With the mBBr-labeled trans-
porter, we utilized mBBr fluorescence, which increases (by
⬃30%) upon TPP binding (Fig. 3C). Notably, by measuring
mBBr fluorescence, we could specifically examine the labeled
protein molecules because unlabeled molecules are invisible.
The results of these studies show that both Cm binding and
mBBr labeling increase the affinity for TPP by ⬃2.5-fold and
that Cm has no influence on the mBBr-labeled protein (Fig. 3, B
and D), consistent with the results of direct binding experi-
ments (Fig. 2B). Thus, affinity measurements imply that mBBr
labeling affects MdfA by a mechanism similar to that of Cm
binding. Because mBBr and Cm are structurally dissimilar (see
Fig. 5A), it seems unlikely that the stimulation is mediated
through direct interaction of Cm or mBBr with TPP. Instead,
the increased TPP affinity probably results from a conforma-
tional change in MdfA that is induced by Cm binding or mBBr
labeling.
The question of whether mBBr must be covalently bound to
induce stimulation of TPP binding was investigated utilizing a
functional Cys-less version of MdfA (6). Because this mutant
does not contain cysteines, it cannot be labeled by mBBr. Fig. 3E
shows that free mBBr did not stimulate TPP binding to Cys-less
MdfA. In contrast, the free compound inhibited TPP binding,
suggesting that mBBr and TPP might compete. Direct binding
competition experiments revealed a k
I
of ⬃34
␮
Mfor mBBr
(Fig. 3E). Thus, for mBBr-induced stimulation of TPP binding
to occur, mBBr must be covalently attached to residue A147C.
Moreover, saturating the transporter with free mBBr did not
prevent Cm from stimulating TPP binding to Cys-less MdfA
(Fig. 3F), indicating that free mBBr interacts with a site that
differs from position 147. Therefore, we conclude that the Cm-
mimicking effect (Fig. 2B) is forced by covalent attachment of
mBBr at position 147.
Substrate-induced Conformational Changes Measured by
Limited Proteolysis—Previous studies (9) and those presented
above suggest that MdfA responds to Cm or covalently bound
mBBr by changing its conformation. To examine this notion
further, we utilized a limited proteolysis approach in which
detergent-solubilized MdfA was proteolyzed by PK in the
absence or presence of substrates or covalent modifications.
The PMSF-sensitive cleavage of Cys-less MdfA by PK pro-
ceeded through several steps (Fig. 4A). The initial cleavage was
relatively rapid (completed by ⬍10 min) and resulted in a ⬃10%
shorter protein, as judged by its mobility in SDS-PAGE (Fig. 4A,
lane 3). This product was designated CF1 (cleavage fragment 1)
(Fig. 4A). The cleavage event that followed was slower (hours)
and generated a shorter proteolytic product, designated CF2
(Fig. 4A,lanes 4–11). The addition of the substrate TPP
changed the proteolysis pattern by significantly increasing the
amount of CF2, with no effect on the amount of CF1 (Fig. 4A,
lane 5), indicating that TPP affects the availability of proteolytic
sites for PK in CF2. This effect most likely results from a sub-
strate-induced conformational change in MdfA (see “Discus-
sion”). Kinetic trials showed that TPP increased the accumula-
tion of CF2 without affecting the rate of CF1 degradation (Fig.
4B, compare lanes 1–7 with lanes 8–14), implying that TPP
binding stabilizes CF2 against cleavage by PK. Notably,
although both CF1 and CF2 were sometimes affected by sub-
strates and chemical modifications, we focused on the behavior
of CF2 because the appearance and disappearance of CF1 were
less reproducible. To assess the specificity of this phenomenon,
we tested how other substrates affect the proteolysis of MdfA
by PK (Fig. 4A). The results show that Cm, ethidium, and tet-
racycline had similar but more moderate effects on the prote-
olysis compared with TPP (lanes 6 –8). In contrast, compounds
FIGURE 3. Comparison of the effects of mBBr and Cm on TPP binding to
MdfA. Aand C, effect of saturating TPP concentrations on the intrinsic fluo-
rescence of unlabeled MdfA-A147C (A) or the mBBr fluorescence of the mBBr-
MdfA-A147C (C). B, TPP binding by unlabeled MdfA-A147C, as measured by
intrinsic fluorescence with and without Cm. D, TPP binding by mBBr-MdfA-
A147C, as measured by mBBr fluorescence with and without Cm. The exper-
iment was performed in triplicate, and the data were fitted to a binding equa-
tion using nonlinear regression software. The indicated dissociation
constants were determined. Eand F, direct measurements of [
3
H]TPP binding
to Cys-less MdfA in the presence of increasing mBBr concentrations (E) and in
the presence or absence of 500
␮
MCm, mBBr, or excess cold TPP (F).
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that are not substrates, such as lactose, spectinomycin, and nali-
dixic acid, did not have any effect compared with the control
sample with no addition (compare lanes 9–11 with lane 4).
Thus, the apparent stabilization of CF2 is mediated by various
substrates that may influence the conformation of MdfA
similarly.
Next, we tested the impact of TPP and Cm on the PK-cata-
lyzed cleavage of mutant MdfA-A147C and a similar picture
emerged, with both substrates stabilizing the CF2 form: TPP
strongly and Cm moderately (Fig. 4C, compare lanes 3 and 4
with lane 2). Interestingly, however, when MdfA-A147C was
labeled by mBBr, the covalent modification per se influenced
CF2 cleavage just like substrates (Fig. 4C,lane 6), reinforcing
the notion that mBBr labeling affects MdfA by inducing a sub-
strate-like conformational change. The addition of Cm or TPP
to mBBr-MdfA-A147C did not significantly improve the stabil-
ity of CF2 (Fig. 4C,lanes 6– 8). Notably, the effect of mBBr on
the proteolysis was more similar to the effect of TPP compared
with Cm. Taking into account the results of the TPP binding
experiments (Figs. 2 and 3), we suggest that both Cm binding
and the covalent attachment of mBBr at position 147 induce a
high TPP affinity conformation in MdfA. The PK cleavage
experiments suggest that the effect of Cm on MdfA-A147C is
moderate compared with that of TPP or covalently bound
mBBr, possibly reflecting a reduced affinity or response of this
mutant to Cm. This finding is also in line with the reduced
stimulation imposed by Cm on TPP binding to this mutant as
compared with Cys-less MdfA (compare Fig. 2Bwith Fig. 3F),
suggesting, as noted above, that the A147C mutation itself par-
tially disrupts the interaction of MdfA with Cm (6).
In direct binding assays, we observed that free mBBr has an
inhibitory effect on TPP binding to Cys-less MdfA (Fig. 3E),
indicating that it might be a substrate and that in its free form it
occupies a site that differs from 147. To test this notion further,
we studied the effect of free mBBr on the proteolysis of the
nonreactive Cys-less transporter. The results indicate that free
mBBr also stabilizes the CF2 fragment against PK (Fig. 4D),
lending support to the suggestion that it is a true substrate of
MdfA.
The Substrate-like Conformational Response to Modification
at Position 147 Is Not Restricted to mBBr—Our results suggest
that forced attachment of mBBr in the putative Cm-binding site
triggers a substrate-induced conformational change in MdfA.
Can this effect also be reproduced with other reagents? To
address this question, we characterized the conformation of
MdfA-A147C after modification by various cysteine-reactive
compounds. We chose six structurally diverse sulfhydryl
reagents that do not resemble known MdfA substrates (Fig.
5A). None of the reagents significantly affected the TPP binding
activity of the nonreactive Cys-less MdfA (Fig. 5B). Similarly,
although the PK-produced fragment CF2 of Cys-less MdfA is
slightly more stable than that of MdfA-A147C, no further sta-
bilization was induced by the free sulfhydryl reagents compared
with TPP- or the Cm-induced CF2 stabilization (Fig. 5C, com-
pare lanes 4–9 with lanes 2 and 3). Next, the reagents were
covalently bound to MdfA-A147C (Fig. 5D), and various effects
of the modifications on TPP binding were clearly evident (Fig.
5E). NEM induced a Cm-like stimulation of [
3
H]TPP bind-
ing, demonstrating that such a conformational response to
modifications at position 147 is not unique to mBBr, despite
the structural difference between mBBr and NEM. In con-
trast, four of the other reagents (AMS, MTSES, MTSEA, and
MTSMT) inhibited TPP binding, whereas the smallest rea-
gent, IAA, did not have any effect. The results also clearly
show that Cm did not stimulate TPP binding to any of the
modified proteins (Fig. 5E). This is not surprising, in light of
the suggestion that residue 147 is important for Cm recog-
nition by MdfA and that modifications at this position by
mutagenesis (6) or chemically (this study) might disrupt Cm
recognition by the transporter.
The stimulatory effect of Cm and covalently bound mBBr or
NEM on TPP binding suggests an allosteric mechanism, in
agreement with the notion that residue 147 is not localized at
the TPP-binding site. Therefore, the observed inhibitory effect
FIGURE 4. Proteolysis of MdfA by PK. A,3
␮
g of Cys-less MdfA were incu-
bated in the absence (lane 1) or presence of PK without or with various com-
pounds. At the indicated times, PMSF was added to stop the reaction. CF1 and
CF2 designate the cleavage fragments that were observed on Coomassie-
stained SDS-PAGE. Time points at 0 and 10 min indicate the fast part of the
cleavage kinetics and are shown as controls. B, kinetics of proteolysis of Cys-
less MdfA in the absence (lanes 1–7) or presence (lanes 8–14) of TPP. At the
indicated times, the reaction was stopped by PMSF. C, proteolysis of MdfA-
A147C and mBBr-MdfA-A147C by PK in the absence or presence of TPP or Cm.
D, proteolysis of Cys-less MdfA by PK in the absence or presence of TPP, Cm,
or free mBBr.
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of certain modifications (AMS, MTSES, MTSEA, and MTSMT,
⬃50– 80% decrease in TPP binding), at this position, on TPP
binding to MdfA is somewhat puzzling (Fig. 5E). Previous stud-
ies demonstrated that positively charged substrates, such as
TPP, form electrostatic interactions with the negatively
charged Glu
26
of MdfA (14, 15), pinpointing a probable binding
site for TPP. In the structural model (Fig. 1), Glu
26
and Ala
147
are located relatively close to each other on opposite faces of the
FIGURE 5. Effects of various sulfhydryl reagents on Cys-less MdfA or MdfA-A147C. A, structures of Cm, TPP, and the tested sulfhydryl reagents shown as
adducts. P-s indicates the sulfur atom of the cysteine residue of the protein. B,[
3
H]TPP binding by Cys-less MdfA in the presence of substrates or free sulfhydryl
reagents. C, proteolysis of Cys-less MdfA by PK in the presence of substrates or free sulfhydryl reagents. The experiment was performed as described in the
legend to Fig. 4 (for 24 h). D, modification of MdfA-A147C by the indicated sulfhydryl reagents and assessment by allowing all purified adducts to react with
Mal-PEG (see the legend to Fig. 2A). E,[
3
H]TPP binding by MdfA-A147C either unmodified or modified by the indicated reagents with or without Cm or excess
of cold TPP. F, proteolysis of unmodified or modified MdfA-A147C versions by PK in the absence or presence of Cm. 10-min time points indicate the fast part of
the cleavage kinetics and are shown as controls.
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putative substrate recognition pocket, suggesting that the Cm
and TPP-binding sites might not be very far apart. Accordingly,
it is possible that certain modifications partially hinder TPP
accessibility and thus prevent detecting possible conforma-
tional effects that might have been triggered by the modifica-
tions. Therefore, we investigated this question by limited
proteolysis.
Fig. 5Fshows the results of limited proteolysis of various
adducts of MdfA-A147C, which can be classified into three
groups. NEM and IAA had a moderate substrate-like stabilizing
effect on CF2; MTSMT and MTSEA stabilized CF2 more sig-
nificantly; MTSES and AMS had no effect on CF2 (Fig. 5F,lane
2in each panel). Notably, unlike mBBr, which exhibited a
strong TPP-like effect (Fig. 4C,lane 6), the effects of NEM, IAA,
MTSMT, and MTSEA on proteolysis were quantitatively more
similar to that of Cm (Fig. 5F,left panel,lane 3, compare with
lane 2 in every the other panel). The addition of Cm could
hardly further stabilize CF2 in these samples, in agreement with
the diminished effect of Cm on TPP binding to these adducts
(Fig. 5E). In conclusion, with the exception of AMS and
MTSES, various dissimilar compounds are able to induce Cm-
like conformational changes in MdfA when attached covalently
to residue A147C. These results suggest that the Mdr recogni-
tion pocket of MdfA is conformationally sensitive to the pres-
ence of various compounds, such that once a molecule is
bound, be it a real substrate or a synthetic covalent attachment,
it might induce a substrate-like conformational change in the
transporter. Therefore, the Cm-like conformational change in
MdfA appears to be induced promiscuously.
Conformational Effects of Cysteine Modifications at Other
Locations in MdfA—To test whether the proposed promiscuity
in inducing conformational changes is restricted to modifica-
tions in a particular site in the recognition pocket of MdfA
(position 147), we examined two other single cysteine mutants:
(i) MdfA-V335C, in which the engineered cysteine is inside the
putative substrate binding pocket, according to previous
genetic and biochemical data (6, 16) and the structural model;
and (ii) MdfA-G410C, containing a cysteine in the nonessential
C terminus of MdfA (17), outside of the putative binding pocket
(Fig. 1). Initially we examined whether the mutant proteins and
their covalent adducts are functional. Direct [
3
H]TPP binding
experiments showed that both mutants could bind TPP to var-
ious extents and were stimulated by Cm (Fig. 6A). Modification
of the C-terminal residue G410C by NEM or MTSEA did not
affect TPP binding or its stimulation by Cm, consistent with its
proposed location in a nonessential part of the protein outside
of the Mdr recognition pocket. Notably, however, mBBr
covalently bound at residue G410C inhibited TPP binding to
some extent, most likely because it is a substrate (Figs. 3Eand
4D) and is somehow able to enter the recognition pocket even if
attached to the C-terminal tail of MdfA. Experimental attempts
to test this hypothesis are underway. In contrast to MdfA-
G410C, modification of MdfA-V335C by either mBBr or
MTSEA had a strong inhibitory effect on TPP binding, whereas
NEM had a small stimulatory effect. Thus, it appears that mod-
ifying V335C affects the interactions of MdfA with TPP, in
agreement with the proposed role of residue Val
335
in substrate
binding (6, 16). Interestingly, however, none of the modifica-
tions in residue V335C prevented the Cm-induced stimulation.
Thus, unlike Ala
147
, residue Val
335
does not appear to be critical
for the interaction with Cm or for the conformational response
to Cm, suggesting that Val
335
represents another face of the
drug-binding pocket.
We then investigated how the substrates and the chemical
modifications influence the conformation of MdfA-G410C and
MdfA-V335C by utilizing the limited proteolysis assay. Almost
all of the modified MdfA mutants responded to Cm and TPP, as
judged by the stabilization of CF2 against proteolysis by PK (Fig.
6, Band C,lanes 3 and 4). A notable exception is the MTSEA-
modified MdfA-V335C, which was impaired in TPP binding
according to both the direct binding assay (Fig. 6A,left panel)
and the limited proteolysis assay (Fig. 6B,third panel from the
left,lane 4). This adduct, however, did respond to Cm, as shown
both in the TPP binding assay where Cm stimulated binding
(Fig. 6A,left panel) and in the limited proteolysis assay where
FIGURE 6. Effects of sulfhydryl reagents covalently bound to MdfA via
residue V335C or G410C. MdfA constructs harboring a single cysteine at
position 335 (V335C) or 410 (G410C) were modified by NEM, MTSEA, or mBBr
or were left unmodified. The proteins were then purified and assayed as fol-
lows. A,[
3
H]TPP binding without or with Cm or excess of cold TPP, as indi-
cated. Band C, proteolysis of unmodified or modified V335C-MdfA (B) and
G410C-MdfA (C) by PK in the absence or presence of Cm or TPP (see the
legend to Fig. 4). 10-min time points indicate the fast part of the cleavage
kinetics and are shown as controls.
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Cm stabilized the CF2 fragment of MTSEA-modified MdfA-
V335C (Fig. 6B,third panel from the left,lane 3). Additionally,
we observed that modification of residue V335C by either
mBBr or NEM affected the cleavage of MdfA by PK in a sub-
strate-like manner, by stabilizing CF2 (Fig. 6Bcompare lane 2
in all panels), whereas modifying residue G410C did not have
this effect (Fig. 6C, compare lane 2 in all panels). Thus, certain
reagents attached to the putative recognition pocket (position
335) trigger a substrate-like conformational effect, whereas
modifying a site outside the pocket (position 410) did not influ-
ence the conformation of MdfA. Overall, although the V335C
site differs from A147C in its response to Cm binding, in both
sites modification leads to a conformational change. Another
property shared by these positions is their location in the sub-
strate-binding pocket, as inferred from previous experiments
showing that: (i) mutations in these positions modulate the
activity of MdfA toward certain drugs and (ii) substrate binding
protects MdfA-A147C and MdfA-V335C against modification
by sulfhydryl reagents (6, 16).
In conclusion, the promiscuous induction of conformational
changes is not restricted to modifications at position 147. We
propose that the Mdr recognition pocket of MdfA is generally
conformationally sensitive and that this may explain how many
dissimilar substrates are able to induce similar conformational
changes upon binding, a prerequisite for Mdr transport.
DISCUSSION
In this study, we utilized purified, detergent-solubilized
MdfA to investigate how this secondary transporter responds
conformationally to substrate binding. The results show that
conformational changes in MdfA are generated promiscuously:
(i) dissimilar substrates induce similar conformational changes
in MdfA and (ii) reagents that are not chemically related to
MdfA substrates induce substrate-like effects, provided that
they are forced to bind at the putative Mdr recognition pocket
of the transporter. A possible explanation for these phenomena
is that MdfA has a sensitive conformational switch in the
pocket that can be triggered by either substrate binding or
chemical modifications.
Mdr transporters from various families were shown to con-
tain multiple drug interaction sites, and these sites are often
allosterically linked (18, 19). In MdfA, binding of Cm was
shown to increase the binding affinity for TPP (9). In theory,
such stimulation of binding could result either from direct
interaction between simultaneously bound substrates or from
substrate-induced conformational changes that influence the
interaction of the transporter with a second substrate. Our
studies show that a Cm-like stimulatory effect on TPP binding
affinity can also be exerted by two Cm-unrelated compounds,
mBBr and NEM, but only when covalently attached to MdfA-
A147C. Considering the structural differences between these
compounds and between them and Cm, a conformational
change is a possible explanation for this effect, and this notion
gains further support from proteolysis trials. In addition, these
results suggest that Ala
147
constitutes part of the Cm interac-
tion site in MdfA, in agreement with previous genetic and bio-
chemical studies (6). Indeed, the location of Ala
147
in the three-
dimensional structural model of MdfA (Fig. 1) suggests that it
lines the putative Mdr recognition pocket.
Mdr transporters belong to structurally diverse families of
transport proteins. This implies that perhaps the Mdr transport
function evolved multiple times during evolution (20). Intrigu-
ingly, however, Mdr transporters from different families share
similar characteristics (1). For example, the mechanism under-
lying Mdr binding is thought to be universal, mainly based on
hydrophobic interaction with substrates (1, 3, 21). Additionally,
transporters from different families were shown to extract sub-
strates not only from the aqueous environment (22–24) but also
directly from the membrane (18, 25, 26). Thus, Mdr transport-
ers from various families cope with common challenges by
adopting similar strategies (4). Better understanding of how
Mdr transporters respond conformationally to dissimilar sub-
strates in a transport-competent manner would add mechanis-
tically important clues to how dissimilar compounds are
actively exported by such transporters. For example, in a P-gly-
coprotein mutant that is defective in biogenesis, it has been
reported that mutations in the binding site improve protein
maturation and that this effect can also be achieved by sub-
strates (27–32). Thus, binding site modifications also have sub-
strate-mimicking effects in this ATP-binding cassette Mdr
transporter. However, the question of whether promiscuously
triggered conformational responses represent a phenomenon
common to Mdr transporters in general remains to be
investigated.
The two largest families of Mdr transporters (major facilita-
tor superfamily and ATP-binding cassette) consist mainly sub-
strate-specific transporters. This implies that the molecular
and structural mechanisms underlying Mdr and substrate-spe-
cific transporters might be similar. One property likely to be
shared by both Mdr and substrate-specific transporters is their
ability to conformationally respond to substrate binding (5, 6,
33) for efficient coupling. This feature suggests that the multi-
specificity of Mdr transporters is reflected in their ability not
only to bind dissimilar substrates but also to generate trans-
port-competent conformational responses to these com-
pounds. This question was partially addressed in this study.
Utilizing direct and indirect measurements of substrate
binding and limited proteolysis, we could detect a substrate-
induced change in MdfA, which leads to a conformation having
high affinity for TPP and resistance to cleavage by PK. In prin-
ciple, substrates can protect against proteolysis by directly
masking potential cleavage sites. However, we favor the possi-
bility that the effects described here are conferred by induced
conformational changes for the following reasons: (i) different
substrates that bind to different sites have similar effects on the
proteolytic profile (Fig. 4A); (ii) modifications in two different
locations of the putative Mdr recognition pocket of MdfA had a
similar stabilizing effect against PK (Figs. 4C,5F, and 6); and (iii)
modification of a single cysteine can either have a stabilizing
effect or not, depending on the attached compound (Figs. 5E
and 6).
The precise role of the observed conformational change
remains to be shown. Nevertheless, this change is probably of
functional relevance because it is induced by various substrates
of the transporter and not by compounds that are not sub-
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strates (unless covalently attached at the substrate-binding
site). Substrate-induced conformational changes are critical for
the transport mechanism, as shown for well characterized
transporters (34, 35), and our results suggest that with MdfA,
dissimilar substrates induce similar conformational changes
and thus might share the same transport mechanism.
The observation that even nonsubstrate compounds can
trigger a substrate-like conformational change when forced to
bind to the putative Mdr recognition pocket of the transporter
is intriguing. We propose that this phenomenon reflects an
inherent property of Mdr transporters, which evolved in such a
manner that enables them to respond to many structurally
unrelated stimuli in the pocket. This property represents an
additional level of multispecificity in MdfA (36) and suggests
that functional conformational changes can be triggered
promiscuously.
Acknowledgments—We thank Marija Jankovic and Philip Hu for help
in the initial characterization of modified MdfA-A147C.
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Conformational Switch in MdfA
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and Eitan Bibi
Nir Fluman, Devora Cohen-Karni, Tali Weiss
MdfA
the Secondary Multidrug Transporter
A Promiscuous Conformational Switch in
and Biogenesis:
Membrane Transport, Structure, Function,
doi: 10.1074/jbc.M109.050658 originally published online October 5, 2009
2009, 284:32296-32304.J. Biol. Chem.
10.1074/jbc.M109.050658Access the most updated version of this article at doi:
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