From the Centre for Infectious Diseases, Wolfson
Research Institute, University of Durham, Queen's Campus,
Stockton-on-Tees, TS17 6BH, § Division of Infection and
Immunity and
Division of Biochemistry and Molecular Biology,
Institute of Biomedical and Life Sciences and ¶ Department of
Chemistry, University of Glasgow, Glasgow G11 6NU, and the
** National Center for Macromolecular Hydrodynamics, School
of Biosciences, University of Nottingham, Sutton Bonington LE12
5RD, United Kingdom
Received for publication, September 16, 2002, and in revised form, December 6, 2002
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ABSTRACT |
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Many pathogenic Gram-negative bacteria possess
tripartite transporters that catalyze drug extrusion across the inner
and outer membranes, thereby conferring resistance. These transporters
consist of inner (IMP) and outer (OMP) membrane proteins, which are
coupled by a periplasmic membrane fusion (MFP) protein. However, it is not know whether the MFP translocates the drug between the membranes, by acting as a channel, or whether it brings the IMP and OMP together, facilitating drug transfer. The MFP EmrA has an elongated periplasmic domain, which binds transported drugs, and is anchored to the inner
membrane by a single A major mechanism of resistance in pathogenic bacteria is the
extrusion of antibiotics from the cell. Gram-negative bacteria possess
tripartite transport systems for translocating drugs across both the
inner membrane (IM)1 and the
outer membrane (OM). This system consists of inner and outer membrane
proteins, which translocate drugs across their respective membranes but
are coupled by a periplasmic protein (1). The periplasmic domain of
this protein is apparently anchored to the IM via either a lipid moiety
or an The structures of two of the components of such a tripartite complex,
the OMP TolC (4) and the IMP AcrB (5), have recently been determined by
x-ray crystallography (4). Both TolC and AcrB crystallize as trimers.
The three TolC molecules are structured into a 140-Å cylindrical
channel with a 35-Å internal diameter. The OM end of the structure is
open, providing solvent access, but the periplasmic end tapers to a
virtual close. The structure can be divided into two major domains: an
OM Interestingly, MFPs are predicted to have a structure that resembles
TolC; the N- and C termini of MFPs are proposed to fold into a
flattened This type of transport system is clearly of considerable scientific and
medical interest, because our knowledge of them is rudimentary, and
their study is likely to have medical benefits, because they confer
drug resistance and only effect transport in bacteria. For this study,
our aim was to characterize EmrA, the MFP of a multidrug transporter
from Escherichia coli (10), as a structural and functional
paradigm for the elucidation of the properties of a number of
IMP-MFP-OMP transport systems and to address the central question of
the role of the MFP in drug translocation. The EmrAB transporter is
composed of EmrB, a putative 14-helix multidrug H+
antiporter belonging to the major facilitator (MF) superfamily (11),
and the MFP EmrA. EmrA is predicted to have a short N-terminal cytoplasmic domain, a single transmembrane helix, and a large periplasmic domain. The EmrAB proteins are thought to provide a
continuous pathway across the bacterial membranes by operating in
conjunction with TolC (1). Underscoring the medical importance of this
system, homologues of EmrA and B have been found in human pathogenic
bacteria such as Vibrio cholerae (12),
Neisseria gonorrhoeae (13), Strenotrophomonas
maltophilia (14), and Campylobacter jejuni (15). Also
the genome sequences of Bacillus subtilis, Haemophilus
influenzae, Neisseria meningitidis, Bordatella pertussis, Rickettsia prowazeki, and Yersinia
pestis indicate that they possess related systems.
Bacterial Strains and Plasmids--
The pGEM-Teasy (Promega)
plasmids bearing the emrA inserts were propagated in
NovaBlue E. coli cells (Novagen). The pET21 (Novagen)
plasmid constructs bearing emrA inserts were propagated in
E. coli strain C41, a derivative of BL21 (16), and that
bearing the hmrA insert was propagated in BL21*
(Invitrogen). A Overexpression and Purification of His-tagged EmrA
Proteins--
Chromosomal DNA from E. coli strain DH5
The pET constructs were used to transform E. coli strain
C41, providing expression of the His6-tagged proteins. A
single colony of C41/pET-EmrA was used to inoculate 5 ml of
tryptone-yeast extract/50 µg ml
Each pET-EmrA construct was tested for expression in the cytoplasm,
inner membrane, and periplasm by purification. Proteins were released
from the periplasm by cold osmotic shock of the cells (18), and the
protein extract was treated according to the procedure adopted for the
purification of soluble EmrA proteins but maintaining the proteins in
glycerol-containing buffers. Proteins were quickly frozen and stored at
Overexpression and Purification of the His-tagged HmrA
Protein--
H. influenzaeRd, KW20, genomic DNA was
obtained from the American Type Culture Collection and used to PCR
clone the periplasmic domain of HmrA, the H. influenzae
homologue of EmrA, with the primers
5'-CACCATGTTTGAAGAAACAGAAGATGCTTATGTGG-3' and
5'-ATGGCTGTTTTGCTGAATGATAGATTC-3'. The resulting product was cloned
into the pET101/D-TOPO (Invitrogen) expression vector
giving the pHmrA-6H plasmid, which was used to transform E. coli TOP10 cells, for overexpression of HmrA-(48-390). Automated
DNA sequencing of the plasmid confirmed the sequence and translation
frame as correct. pHmrA-6H was used to transform E. coli
BL21* (Invitrogen), which was used for all subsequent protein production.
BL21*/pHmrA-6H cells were grown at 37 °C in a 10-ml LB starter
culture containing 100 µg/ml carbenicillin from a single colony picked from a fresh agar plate. When the cells were just visible, 1 ml
of starter culture was used to inoculate 1 liter of LB containing 100 µg/ml CB, which was grown to an A600 of 0.4 at
37 °C with shaking at 200 rpm. Cells were induced with 1 mM isopropyl-1-thio- Construction of pUC-EmrAB, pUC-EmrA-(49-390)B and
pUC-HmrAB--
The emrA and emrB genes were
amplified by PCR using the forward and reverse primers
5'-GAATTCGAGCGCAAATGCGGAGACTC-3' and
5'-GAAGCTTAGTGCGCACCTCCGCC-3', respectively, to introduce
EcoRI and HindIII sites (underlined) at the 5'
and 3' ends of the amplified DNA, which was purified and ligated into
pGEMT-Easy (Promega). The emrAB genes were rescued from
pGEM-Teasy by restriction digest with EcoRI and
HindIII, ligated into
EcoRI/HindIII-digested pUC to create pUC-EmrAB,
and transformed into E. coli strain N43. Similarly, the
pUC-EmrA-(49-390)B construct was made using the forward primer
5'-GAATTCGAAGAAACCGATGACGCATACG-3', so that the expressed EmrA lacked
the first 48 amino acids. The constructs were checked by automated DNA
sequencing. The hmrA and hmrB genes were
amplified by PCR using the forward and reverse primers
5'-GAATTCTGACGCAAATTGCAACT-3' and 5'-GAAGCTTAATGCTGAGTACC AAA-3',
respectively. The PCR product was purified and ligated into pGEM-Teasy
(Promega). The hmrAB genes were rescued from pGEM-Teasy by
restriction digest with EcoRI, ligated into
EcoRI-digested, alkaline phosphatase-treated pUC to create
pUC-HmrAB, and transformed into E. coli (NovoBlue,
Invitrogen). Automated DNA sequencing was used to identify a plasmid in
which the hmrAB genes were correctly orientated, which was
then used to transform strain N43.
MIC Measurements--
MICs were measured according to the
microdilution broth method established by the National Committee for
Clinical Laboratory Standards (19). Briefly, a single colony was picked
from an LB/ampicillin plate, used to inoculate 20 ml LB/ampicillin
medium, and grown to an A600 of 0.1. Cells were
transferred to a microtiter plate and mixed with serial dilutions of
the drug to be tested. Bacterial growth was monitored after an 18-h
incubation at 37 °C.
Protein Concentration and Gel Analysis--
Protein
concentrations were determined by the BCA assay using a kit from Pierce
with bovine serum albumin as standard. Proteins were separated by
SDS-PAGE on 4-12% or 12% polyacrylamide gradient gels (NuPAGE gels
and MES or MOPS buffer; Novex) and stained with Coomassie Gelcode
BlueTM (Pierce). Protein samples for SDS-PAGE were mixed
with the loading buffer at room temperature to avoid any potential
problems due to protein aggregation, which can occur when samples are
boiled. Native PAGE (7.5% (BioRad) polyacrylamide gels and
Tris-glycine buffer (pH 7.5) run for 16 h at 50 v) was also
used to separate proteins.
Gel Chromatography--
EmrA proteins were subjected to gel
chromatography on a Superdex 200 column run on an AKTA purifier
(Amersham Biosciences) automated chromatography system.
EmrA-(1-390) (1.3 mg ml Western Blotting and N-terminal Sequencing--
EmrA-(1-390)
monomers and dimers were separated by gel chromatography on a Superdex
200 column, resolved by 12% SDS-PAGE, transferred to polyvinylidene
difluoride membrane and either immunoblotted with monoclonal
anti-polyhistidine antibodies (Sigma) or N-terminal sequenced (Alta
Bioscience, University of Birmingham, UK). The predicted amino acid
sequence of the EmrA-(2-390)-(His)6 fusion protein
(i.e. termed EmrA-(1-390)) was
MASMTGGQQMGRDP-EmrA-(2-390)-LEHHHHHH, with residues 2-12 constituting
an immunogenic T7 tag. N-terminal sequencing of the EmrA-(1-390) gave
the first 11 and 10 residues of the T7 tag for the monomer and dimer, respectively.
Spectroscopic Analyses--
CD spectra were recorded on a Jasco
J600 spectrometer at 20 °C using protein samples dialyzed into 50 mM Tris-acetic acid (pH 8.0), 200 mM NaF. The
percentage of secondary structure was predicted from the CD spectra
using the program SELCON (20).
Fluorescence measurements were made in a Jasco FP750 fluorimeter at
20 °C. Tryptophan fluorescence was excited at 292.5 nm, and the
emission wavelength was scanned between 300 and 400 nm. For titrations
of EmrA-(49-390), KI was added from a 5 M stock solution
to 2 ml of 1.25 µM protein in 50 mM Tris-HCl
(pH 8.5), 200 mM NaCl. For KI titrations in the presence of
drugs, the drug concentration was set to the maximum possible
(e.g. 39 µM FCCP, 48 µM CCCP, 19 µM DNP, 20 µM nalidixic acid, and 53 µM chloramphenicol) that would not reduce the protein
fluorescence by more than 50% because of its inner filter effect. In
the case of nalidixic acid, the pH of the protein solutions was tested
to ensure that the small volume of acid added had not perturbed the pH.
The protein fluorescence and KI concentration were corrected for the
dilution effect. HmrA-(48-390) was routinely used at a concentration
of 6 µM because this gave a fluorescence equivalent to
1.25 µM EmrA-(49-390), but using the proteins at
equivalent concentrations did not affect the titration curves. The FCCP
titration curve for EmrA was fitted to an equation with hyperbolic and
linear functions by nonlinear regression using the program SigmaPlot
(Jandel Scientific): y = A Analytical Ultracentrifugation--
Sedimentation velocity and
sedimentation equilibrium measurements were made with an Optima XL-A
analytical Ultracentrifuge (Beckman). Sedimentation was performed at
45,000 rpm in double sector cells at 20 °C, and the data were
analyzed using DCDT+ software, version 1.12 (21). We measured the value
of s for different protein concentrations in the range of
0.5-1 mg/ml in 40 mM potassium phosphate (pH 8.0), 400 mM KCl and extrapolated the data to zero protein
concentration. Sedimentation equilibrium experiments were performed at
15,000, 18,000 and 20,000 rpm at 20 °C, and the data were analyzed
using the manufacturer's software (Microcal Origin, version 4.1). The
partial specific volume, Dynamic Light Scattering--
Dynamic light scattering
experiments were performed on a Dynapro 801 (Protein Solutions Inc.)
instrument at 20 °C. Samples were injected at a concentration of 1 mg ml EmrA Has a Periplasmic Domain That Is Anchored to the Membrane by a
Single EmrA Forms Dimers and Trimers--
An analysis of the
EmrA-(1-390) protein by SDS-PAGE (4-12% polyacrylamide gel, MES
buffer) revealed two predominant bands, one migrating close to the
expected molecular mass of 45.1 kDa and the other with an apparent
molecular mass of about 60 kDa (Fig. 2A, lane 5). Similarly,
two predominant bands were apparent for the truncate EmrA-(15-390),
but both bands ran slightly faster than the corresponding bands for
EmrA-(1-390) (Fig. 2, A, lane 4, and
B). This suggested that the higher molecular mass bands were
dimers of EmrA that ran at a lower than expected molecular mass, as is
usually the case for membrane proteins. However, better resolution of
the dimer molecular mass was obtained under alternate SDS-PAGE
conditions (12% gel, MOPS buffer), indicating that the dimer has a
molecular mass in excess of 64 kDa (Fig. 2B, lane 2). It seems unlikely that these high molecular mass bands are attributable to complexes with other proteins, such as EmrB and TolC,
because their intensity is much greater than would be expected when
considering the wild-type levels of expression of these proteins. Moreover, although EmrA does not possess any cysteine residues, the
putative dimer could be dissociated upon treatment with 4 M
The N Terminus of EmrA Contains a Leucine zipper Motif That Is
Important for Dimerization--
In contrast to EmrA-(1-390), soluble
EmrA-(49-390) migrated as a single band with an apparent molecular
mass of 40 kDa, consistent with the calculated molecular mass of 39.6 kDa (Fig. 2A, lane 2). Furthermore,
EmrA-(49-390) predominantly eluted as a single protein peak from a gel
chromatography column, consistent with its monomeric form (Fig.
2F). The slight shoulders on either side the central protein
peak are probably attributable to small amounts of the dimer and a
lower molecular mass-contaminating protein, which can be detected on
Coomassie-stained gels generally overloaded with EmrA proteins. We note
that the size of the contaminating protein is dependent upon the EmrA
truncate that it accompanies, being larger for EmrA (1-390) than
EmrA-(49-390), suggesting that it is a product of proteolytic
degradation of EmrA. These findings are consistent with the prediction
that residues 23-46 form a membrane-spanning Characterization of the Periplasmic Domain of EmrA--
We used
the secondary structure prediction program Jpred (24), which
indicated that the periplasmic domain of EmrA consists of a large
If EmrA-(49-390) has two distinct domains, an
We decided to test whether EmrA might also be an elongated molecule,
consistent with the predicted structure of a EmrA Is Required for Antibiotic Resistance--
pUC constructs
were used to express EmrA-EmrB and EmrA-(49-390)-EmrA-EmrB in
the E. coli acr deletion strain, N43, which is highly susceptible to a wide range of antibiotics (17). Although full-length EmrA and -B were able to confer resistance to CCCP, FCCP,
DNP, and nalidixic acid but not erythromycin or chloramphenicol, cells
expressing EmrA-(49-390)-EmrA and EmrB were as susceptible to these
agents as control cells transformed with pUC (Table
II). Because the EmrA-(49-390)-EmrA
would not be targeted to the membrane, the data indicate that
resistance cannot be conferred solely by EmrB. We also tested the
homologous genes hmrA and hmrB from H. influenzae for their ability to confer resistance to these agents. Although these genes did not confer resistance to FCCP and CCCP, they
were able to confer resistance to erythromycin, indicating that the Hmr
transport system was functional in strain N43 (Table II). In this
respect, HmrAB is not unique, because expression of VceAB
from V. cholerae has been shown to confer resistance to
drugs, including CCCP, when expressed in a The Periplasmic Domain of EmrA Binds Transported Drugs--
A
potential role for EmrA is in the binding of drugs exiting EmrB for
transfer to TolC. In this case it might be possible to detect the
binding of drugs by fluorescence spectroscopy if any of the four
tryptophan residues present in the periplasmic domain were to
change environment upon drug binding. Unfortunately it is not possible
to monitor the interaction of these drugs with EmrA directly from
changes in the tryptophan fluorescence because they all produce a
significant inner filter effect that obscures any change in tryptophan
fluorescence. However, the exposure of the tryptophan residues of a
protein can be assessed by their accessibility to quenching by
potassium iodide (28). The binding of a drug to EmrA might be detected
as a change in the exposure of the tryptophan residues resulting from a
conformational change. Consequently, as an alternative to direct
measurements of drug induced changes in the tryptophan fluorescence, we
determined the exposure of the tryptophan residues in the absence and
presence of drugs. If the protein is titrated with KI, monitoring the
quench in fluorescence, the data can be analyzed as a Stern-Volmer plot of F0/F versus the KI
concentration, where F0 and F are the
fluorescence of the protein in the absence and presence of KI,
respectively (29). A linear plot is indicative of a single class of
quenchable tryptophan, but a downwardly curved plot indicates that
there are tryptophan residues that differ in their accessibility to KI
(28). As shown in Fig. 4A, the
Stern-Volmer plot for the KI quenching of EmrA-(49-390) was convex to
the abscissa, indicating that the four tryptophan residues present in
EmrA-(49-390) differ in their accessibility to KI. Clearly, in the
presence of the transported drugs FCCP, CCCP, DNP, and nalidixic acid,
the exposure of the tryptophan residues was reduced, indicating that
the binding of these drugs caused a conformational change in EmrA. In
contrast, chloramphenicol, which is not a substrate of the EmrAB
transporter, did not appreciably reduce the exposure of the tryptophan
residues of EmrA. Although it is unlikely that the nonspecific binding of these drugs would cause a conformational change at the
concentrations used, we sought to investigate this possibility using
HmrA. It is reasonable to conclude that the HmrA and -B proteins do not interact with FCCP because they do not confer resistance to this drug.
However, although the Emr and Hmr transporters differ in their drug
specificity, HmrA has 46.2% identity with EmrA, with three of the
tryptophans conserved in the two proteins, suggesting that they will
adopt very similar structures. Consistent with this prediction, EmrA
and HmrA have similar CD spectra (Fig. 3A). Accordingly,
HmrA-(48-390) was titrated with KI in the absence and presence of FCCP
as a control to test for the effects of the nonspecific binding of
FCCP. As shown in Fig. 4B, a significant difference in the
value of F0/F in the absence and
presence of FCCP was apparent only at the highest KI concentration,
consistent with a small nonspecific binding effect. To test whether the
HmrA was correctly folded and capable of binding transported
substrates, the protein was titrated in the presence of erythromycin
and nalidixic acid (Fig. 4C). Interestingly, we found that
HmrA did not behave in an identical manner to EmrA, in that these
substrates caused an increase in the
F0/F ratio for HmrA, indicative of an
increase in the exposure of the tryptophans due to binding of these
substrates. It is possible that this difference results from the
binding of drugs in the vicinity of Trp76 of EmrA that is
not conserved in HmrA.
We used the difference in quenching with 1 M KI, in the
presence and absence of FCCP, to titrate EmrA-(49-390) with FCCP (Fig. 5). For low concentrations of FCCP the
value for F0/F decreased in a
hyperbolic manner, but as the concentration was increased F0/F continued to decrease in an
apparently linear manner. This behavior is consistent with the specific
binding of FCCP at low concentrations but with nonspecific binding
becoming apparent at higher concentrations. Accordingly, we analyzed
the titration curve by nonlinear regression fitting to an equation
incorporating hyperbolic and linear functions; this fitting procedure
indicated a maximal quench in the fluorescence of 8.1 (±2.8) % and a
Kd for the specific EmrA-(49-390)-FCCP complex of
4.2 (±2.6) µM. The EmrE-FCCP complex has a similar
Kd value of 3.0 µM (30). In contrast,
when HmrA-(48-390) was titrated with FCCP, F0/F decreased in a linear manner
with increasing FCCP concentration. This behavior parallels that of
EmrA-(49-390) at high FCCP concentrations and is consistent with the
nonspecific binding of FCCP by HmrA-(48-390) (Fig. 4C). A
similar analysis yielded Kd values of 1.2 (±1.1)
µM and 9.4 (±6.6) µM for the binding of
CCCP and DNP, respectively, to EmrA (data not
shown),3 indicative of a
correlation between the affinity of EmrA for the drug and the
resistance afforded. For example, EmrA binds CCCP with the highest
affinity, and this drug is the best substrate for the EmrAB transport
system as judged by the increase in the MIC upon expression of the
transport system in a The aim of this study was to characterize EmrA, a membrane fusion
protein from a tripartite multidrug extrusion system. By truncating the EmrA protein we were able to show that it is anchored to
the inner membrane by residues 1-59, consistent with the proposal that
residues 23-46 form a membrane-spanning Herein we provide evidence that membrane-bound EmrA forms dimers and
trimers. What is the structural basis for the oligomerization of EmrA?
EmrA is predicted to have an EmrA and TolC are predicted to have similar tertiary and quaternary
structures, an elongated On the basis of the structure of the RND antiporter, AcrB, it has been
suggested that the MFP AcrA binds to a large cleft in the periplasmic
headpiece of AcrB (5). In this position it would overlap the
periplasmic domains of AcrB and TolC, where it could play a role in
recruiting TolC. If this is the case, it brings into question the site
of interaction of EmrA with EmrB, because EmrB is a major facilitator
antiporter (1), which does not possess large periplasmic domains
between helices 1 and 2 and helices 7 and 8. Perhaps the leucine zipper
motif of EmrA is of importance in the EmrA-EmrB interaction, because it
is noticeable that whereas EmrA is anchored by an A plausible model for the structure and function of the EmrB-EmrA-TolC
tripartite transport system might be one in which EmrB and EmrA form a
stable complex, possibly via their membrane-spanning leucine zipper
motifs, positioning the -helix, which contains a leucine zipper dimerization domain. Consistent with CD and hydrodynamic analyses, the
periplasmic domain is predicted to be composed of a
-sheet subdomain
and an
-helical coiled-coil. We propose that EmrA forms a trimer in
which the coiled-coils radiate across the periplasm, where they could
sequester the OMP TolC. The "free" leucine zipper in the EmrA
trimer might stabilize the interaction with the IMP EmrB, which also
possesses leucine zipper motifs in the putative N- and C-terminal
helices. The
-sheet subdomain of EmrA would sit at the membrane
surface adjacent to the EmrB, from which it receives the transported
drug, inducing a conformational change that triggers the interaction
with the OMP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helix. There has been much speculation as to the functional
role of this periplasmic protein, the delineation of which is crucial
to understanding the mechanism of this type of transport system. One
proposal is that it forms a channel between the membranes; but another
suggests that it pulls the membranes together, allowing ligand transfer between the IMP and OMP (2). Because of the latter hypothesis this
periplasmic protein was originally termed a membrane fusion protein
(MFP), but more recently the term dynamic adaptor has been adopted
(3).
-barrel and a periplasmic
-helical barrel. The
-barrel
domain, which provides an essentially open channel through the OM, is
composed of 12
-strands, 4 donated by each TolC molecule, arranged
into a right-twisted barrel. The
-helical domain is a 12-helix
barrel, constructed from long (67 residues) and short (23 and 34 residues) helices, with pairs of the shorter helices stacked to produce
pseudo-continuous helices. The
-helices are further arranged into
coiled-coils, and the mixed
/
structure connecting the shorter
helices forms a belt around the helical barrel. The
-helical barrel
is about 100 Å long, which is close to the lower estimates of the
depth of the periplasmic space at 130 Å, but some estimates put the
depth of the periplasm at 250 Å and beyond the span of TolC (6, 7). The AcrB trimer, which has a jellyfish-like appearance,
comprises a periplasmic headpiece with dimensions of 50 × >100
Å and a transmembrane domain with dimensions of 70 × >80 Å (5). The headpiece, which is formed by protrusions between helices 1 and 2 and helices 7 and 8 of the transmembrane domain, is divided into
two stacked parts, with the upper and lower parts 30 and 40 Å thick,
respectively. Viewed from the side, the upper part has a trapezoidal
appearance, 70 Å wide at the bottom and 40 Å at the top; whereas
viewed from above, the upper part is open like a funnel, with an
internal diameter of 30 Å. This funnel is connected by a pore, located between the headpieces of the three protomers, to a large central cavity at the interface of the headpiece and the transmembrane domains
of the protomers. The three transmembrane domains, each of which is
composed of 12 helices, are arranged into a ring with a 30-Å hole
between them, which might be filled with phospholipids. It has been
proposed that the upper headpiece interacts with TolC (5), with six
vertical hairpins from the AcrAB trimer contacting the six
-helix-turn-
-helix structures of the TolC trimer, to form a
continuous path across the periplasmic space. If this is the case, it
suggests a mechanism in which drugs transported through the
transmembrane domains of AcrB are delivered to the central cavity created at the transmembrane domain headpiece interface, where
they can be shuttled through the headpiece pore and funnel to TolC.
-barrel, with the intervening residues arranged into two
long helices, each of about 60 or more residues, which fold back on one
another to form a coiled-coil (2). Considering those MFPs that utilize
an N-terminal
-helix to anchor them to the IM, this would position
the
-barrel at the IM with the
-helices radiating out across the
periplasm. Furthermore, the ability of MFPs to form stable trimers (8,
9) invites the suggestion that their role is to form a connecting
channel between the IM translocase and TolC. The putative
-barrel of
the MFP could act as the receiver domain for drugs released from the IM
translocase, whereas the
-helices could transiently interact with
TolC. A possible mechanism for this interaction is that the six
-helices of the MFP trimer form a cylinder that inserts into the
closed end of TolC to open it. Considering however that both TolC and MFPs are highly elongated molecules (4, 9) capable of overlapping in
the periplasmic space, a more likely mechanism is for the MFP to
utilize its
-helices to "grab" the outer surface of TolC. There
is a deep cleft within the headpiece of AcrB in which the MFP AcrA may
lie, thereby positioning it to straddle both the periplasmic
domains of AcrB and TolC (5), and biochemical cross-linking studies
have revealed that the MFP-TolC interaction is substrate-induced and
transient (8). On the other hand, the
-domain contains a motif that
resembles the lipoyl domain of enzymes involved in the transfer of a
covalently attached lipoyl or biotinyl moiety between proteins (2). In
such enzymes, this lipoyl domain is usually a flattened
-barrel. The
formation of a similar domain would require the N- and C-terminal
domains of the MFP to interact, which might provide a mechanism for
bringing the two membranes together. However, the dimensions of AcrB
and TolC are sufficient to indicate that they can contact one another
across the periplasm, arguing against a role for the MFP in bringing
the IM and OM together.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
acrA strain of E. coli, termed
N43 (17), was transformed with E. coli emrAB,
NTemrAemrB (i.e. the construct expressed
EmrA-(49-390)), and H. influenzae hmrAB pUC
constructs and used for MIC measurements.
was used as target DNA for amplification of emrA by PCR
using the forward and reverse oligonucleotide primers
5'-GGATCCAGCGCAAATGCGGAGACTCA-3' and
5'-CTCGAGGCCAGCGTTAGCTTTTACGAT-3', respectively. We
incorporated BamHI and XhoI restriction sites (underlined) into these primers to allow fragment ligation into pET21a
to produce construct pET-EmrA-(1-390). Four truncated fragments of
EmrA were generated by PCR, emrA-(15-390),
emrA-(29-390), and emrA-(49-390), with the
forward primers 5'-GGATCCAAGAGCGGCAAACGTAAG-3', 5'-GGATCCC
TCTTTATAATTATTGCCGT-3' and 5'-GGATCCGAAGAAACCGATGACGCATACG-3', respectively, in combination with the reverse primer used to clone emrA-(1-390), whereas emrA-(1-56) was generated
with the forward primer used to clone emrA-(1-390) and the
reverse primer 5'-CTCGAGCGTATGCGTCATCGGTTTCTTC-3'. In each case
BamHI-XhoI restriction fragments were prepared
and ligated into pET21a to produce constructs pET-EmrA-(15-390),
pET-EmrA-(29-390), pET-EmrA-(49-390), and pET-EmrA-(1-56). Each pET
construct was sequenced to ensure its integrity and that it was
in-frame with the T7 and His6 tags.
1 carbenicillin
(CB), grown to saturation at 37 °C, and used to inoculate 0.5 liters
of tryptone-yeast extract/50 µg ml
1 CB. Growth was
continued at 37 °C until reaching an
A600 of 0.4-0.5, at which point 1 mM isopropyl-1-thio-
-D-galactopyranoside/50 µg ml
1 CB was added, and the growth continued overnight
at a reduced temperature of 23 °C. For most preparations 3 liters of
cells (e.g. 6 × 0.5 liters) were cultivated. Cells
were harvested at 8670 × g and washed with TNG buffer
(50 mM Tris-HCl (pH 8.5), 150 mM NaCl, 10%
glycerol). The cell pellet from each 0.5-liter culture was resuspended
in 20 ml of TNG buffer, giving 100 ml for a 3-liter culture, to which
was added 0.5 ml 10 mg ml
1 lysozyme, 0.1 ml 10 mg
ml
1 DNase, and 1 protease inhibitor mixture tablet
(complete, EDTA-freeTM, Roche Molecular Biochemicals). The
cells were disrupted by passage through a cell disrupter (model Z-plus
1.1 kilowatts, Constant Systems) operated at 4 °C. Unbroken
cells and cell debris were cleared from the supernatant at 39,000 × g (20 min, 4 °C), which was then spun at 194,000 × g (1.5 h, 4 °C), separating the soluble protein from
the cell membrane pellet. EmrA-(49-390) was isolated from the
supernatant, EmrA-(1-56), EmrA-(15-390), and EmrA-(1-390) from the
membranes, and EmrA-(29-390) from both the membrane and soluble
fractions. For purification of EmrA-(49-390), 6 ml of suspended
Ni2+-NTA-agarose (Qiagen) was added to one-quarter of the
supernatant and incubated for 1 h at 4 °C. A 1.5-cm-diameter
Econo-columnTM (BioRad) was packed with the
Ni2+-NTA-agarose to a final column volume of about 3 ml and
washed successively with 10, 2.5, and 0.5 volumes of TNG buffer
containing 10, 25, and 50 mM imidazole, respectively,
before elution of the protein under gravity with 400 mM
imidazole in 50 mM Tris-HCl (pH 8.5), 200 mM
KCl (TK). Generally, 5 ml of protein at 15-20 mg ml
1 was
eluted from the column, and then a further 5 ml of protein at 5 mg
ml
1 was eluted, thus giving a yield of about 400 mg of protein from a 3-liter culture. The protein was dialyzed against
TK to remove the imidazole. EmrA-(29-390) was obtained from the
soluble fraction in an identical manner. For EmrA-(1-390),
EmrA-(1-56), EmrA-(15-390), and EmrA-(29-390) (membrane fraction)
purifications, each membrane pellet was solubilized by the addition of
1 volume of 1.5% dodecyl-
-D-maltoside in TKG buffer (TK
plus 10% glycerol) and incubated on ice for 1 h, after which time
9 volumes of TKG was added to reduce the dodecyl-
-D-maltoside concentration to 0.15%, and
solubilized proteins were separated from the membrane debris at
194,000 × g. To 100 ml of supernatant, 4 ml of
suspended Ni2+-NTA-agarose/1 protease inhibitor tablet was
added and incubated for 3 h at 4 °C. A 1-cm-diameter
Econo-column was packed with the Ni2+-NTA-agarose to a
final volume of about 2 ml and washed successively with 10, 2.5, and 2 volumes of TKGN (TKG plus 0.2%
n-nonyl-
-D-maltoside) containing 10, 25, and
50 mM imidazole, respectively. 400 mM
imidazole/TKGN was used to elute the protein under gravity, with the
protein usually occurring in the second and third 1-ml aliquot at a
concentration of 0.5-1 mg ml
1. The protein was dialyzed
against TKGN to remove the imidazole.
80 °C.
-D-galactopyranoside for
3 h at 25 °C and then chilled on ice for 1 h. The cells
were harvested by centrifugation, resuspended in 50 mM
HEPES (pH 8.0), 300 mM KCl, and disrupted with a Cell
Disrupter (Constant Systems). The cell debris was removed by
centrifugation and frozen in five 25-ml aliquots at
80 °C.
HmrA-(48-390) was purified according to the same protocol adopted for
EmrA-(49-390).
1 in TKGN) and EmrA-(49-390)
(3.0 mg ml
1 in TKG) were applied to a Superdex 200 HR
10/30 column equilibrated with PNGL buffer (25 mM sodium
phosphate (pH 8.0), 150 mM NaCl, 10% glycerol, 0.1%
lauryldimethylamine-N-oxide) and eluted at a flow rate of
0.25 ml min
1. It was necessary to utilize PNGL as the
equilibration buffer because the separation of the monomers and dimers
of EmrA was inefficient when TKGN was used as the equilibration buffer,
which however is better for storage of EmrA. Although the retention time for the EmrA-(49-390) monomer appeared to be less than for the
EmrA-(1-390) dimer and monomer, possibly because the proteins adopt
different conformations, the column resolution would be relatively poor
in this range (e.g. between 45 and 90 kDa), and consequently
there would be difficulties in comparing the elution profiles from
different runs. Accordingly, we did not try to estimate the molecular
masses of the proteins from the retention times. Moreover, the proteins
are likely to elute as protein-detergent micelles with a greater
molecular mass than the protein.
[(B × [FCCP]/Kd + [FCCP]) + (C × [FCCP])], where A is the value of
F0/F in the absence of FCCP,
B is the total decrease in the value of
F0/F for concentrations of FCCP that tend toward saturating the hyperbolic component attributed to specific
binding, Kd is the dissociation constant for the
specific EmrA-(49-390)-FCCP complex, and C is the slope of the linear component attributed to nonspecific binding.
, was calculated as
0.736 ml/g from the amino acid sequence of EmrA-(49-390) using
SEDNTERP software (22); this value was used in all calculations.
1. Data analysis was performed using the
manufacturer's software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Helical Domain--
Residues 23-46 of EmrA were
predicted, using the program TMHMM (23), to form an
-helix that
anchors the protein in the periplasm of E. coli.
However, this prediction for EmrA, or for any other MFP, has
not been tested experimentally. To map the domains of EmrA, a number of
emrA constructs were made in pET to overexpress the whole
and truncated portions of EmrA (Fig. 1)
with a His tag to aid in purification (Fig.
2A). To determine the location of each protein,
we attempted their purification from the periplasm milieu, obtained by
osmotic shock of the cells, from the cytoplasmic milieu, obtained as
the soluble fraction after cell disruption, and from inner membranes,
prepared by detergent solubilization of membranes from disrupted
cells. The EmrA-(1-390), EmrA-(15-390),
and EmrA-(1-59) proteins were purified from cytoplasmic membranes,
whereas EmrA-(49-390) was purified as a soluble protein from the
cytoplasm. In contrast, EmrA-(29-390) was obtained from both membranes
(about two-thirds of the total protein) and the cytoplasm (about
one-third of the total protein). None of the proteins was purified from
the periplasmic milieu following osmotic shock. We concluded that EmrA
is anchored to the membrane by residues 15-49.
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Fig. 1.
Schematic diagram of EmrA based upon the
predicted secondary structure and domain mapping experiments.
A, schematic model of the EmrA secondary structure. The
amino acid sequence surrounding the N-terminal -helix that anchors
EmrA to the inner membrane is shown, indicating the positions of the
truncated EmrA constructs (in B) used to map the EmrA
domains. Those residues belonging to a putative leucine zipper motif,
which runs through the
-helix, are underlined and
italic. B, predicted topologies of the truncated
derivatives of EmrA constructed to map the domains of EmrA. The
-helices and
-sheet domains are shown as rectangles
and saw-tooth schematics, respectively. The
letters in the membrane-spanning helix represent the leucine
zipper motif; note that this is truncated in EmrA-(29-390).
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Fig. 2.
EmrA is anchored to the membrane by an
N-terminal -helix that contains a dimerization
domain. A, SDS-PAGE (4-12% polyacrylamide gel, MES
buffer) analysis of the EmrA proteins: EmrA-(49-390) (lane
2), EmrA-(29-390) (lane 3), EmrA-(15-390) (lane
4), EmrA-(1-390) (lanes 5), and EmrA-(1-56)
(lane 7). EmrA-(29-390), EmrA-(15-390), EmrA-(1-390), and
EmrA-(1-56) were isolated from membranes and EmrA-(49-390) from the
cytoplasm, consistent with residues 23-46 forming an
-helical
membrane anchor. EmrA-(15-390), EmrA-(1-390), and EmrA-(1-56), but
not EmrA-(49-390), are shown as forming dimers, demonstrating that
this
-helix contains a dimerization domain. Consistent with the
presence of a functional leucine zipper in EmrA, EmrA-(29-390), which
lacks the first Leu of the putative leucine zipper motif, was
membrane-bound but failed to form dimers. B, SDS-PAGE (12%
polyacrylamide gel, MOPS buffer) analysis of untreated EmrA-(1-390)
(lane 2) and EmrA-(1-390) treated with 4 M
-mercaptoethanol (lane 1), establishing that the higher
molecular mass band is a dimer that dissociates when treated with 4 M
-mercaptoethanol. C, SDS-PAGE (12%
polyacrylamide gel, MOPS buffer) analysis of EmrA-(1-390) eluted from
a nickel-agarose column: lanes 1, 2, and 3 are the second 1-ml fraction after the column is
eluted with 250 mM imidazole (lane 1), the first
1-ml fraction (lane 2), and the pooled second to eighth 1-ml
fractions (lane 3) , respectively, after elution with 400 mM imidazole. The trimer appears concentrated in the first
1-ml fraction eluted with 400 mM imidazole. D, a
native gel (7.5% polyacrylamide, Tris-glycine buffer (pH 7.5), run at
50 v for 16 h) establishing that EmrA-(1-390) forms
oligomers in the absence of SDS (e.g. putative monomer,
dimer, and trimer bands are indicated by arrows).
E and F, separation of EmrA monomers and dimers
by gel chromatography. The elution profiles for EmrA-(1-390)
(E) and EmrA-(49-390) (F) eluted from a Superdex
200 column indicate that whereas EmrA-(1-390) exists in both
monomeric and dimeric forms, EmrA-(49-390) is predominantly monomeric.
G, SDS-PAGE (12% polyacrylamide gel, MOPS buffer) analysis
of protein samples eluted from the Superdex 200 column: EmrA-(49-390)
protein eluting between 10.5 and 11.5 ml (lane 1);
EmrA-(1-390) protein prior to application to a Superdex 200 column
(lane 2); EmrA-(1-390) protein eluting between 11 and 12 ml, which had been incubated with the gel buffer for 20 min at 20 °C
(lane 4) and 75 °C (lane 5) and treated with 4 M
-mercaptoethanol (lane 6); EmrA-(1-390)
protein eluting between 12.5 and 14 ml (lane 8) and the same
protein heated to 75 °C for 20 min in the presence of 4 M
-mercaptoethanol (lane 7). H,
Western blot, using anti-polyhistidine antibodies, of the purified
protein fractions from the Superdex 200 column: the putative
EmrA-(1-390) monomer eluting between 12.5 and 15 ml before (lane
1) and after (lane 2) treatment with 4 M
-mercaptoethanol; and the putative dimer, eluting between 10.5 and
12 ml, before (lane 3) and after (lane 4)
treatment with 4 M
-mercaptoethanol.
-mercaptoethanol, with only the 45-kDa band apparent after treatment
(Fig. 2B, lane 1). Dissociation of the dimer was
presumably due to solvent effects, because the concentration of
-mercaptoethanol required to destabilize the dimer (e.g.
>2 M) was much greater than normally used to reduce
disulfide bridges (e.g. 0.1 M) and the dimer
could also be dissociated with 4 M Me2SO (data
not shown). In some preparations we noted the presence of a third
predominant band with a molecular mass of >100 kDa, suggestive of a
trimer. However, this band was not present in all preparations, just in the most concentrated protein preparations, and it tended to disappear after extensive dialysis. This suggested that the monomer, dimer, and
trimer are in a concentration-dependent equilibrium. To
investigate this possibility further, we introduced a second elution
step into the EmrA-(1-390) protein purification procedure, eluting the
protein from the nickel-agarose column first with 250 and then 400 mM imidazole and analyzing the samples by SDS-PAGE before dialysis. Our rational was that the trimer, which would have three Ni2+-binding sites, would be held more tightly
by the nickel-agarose than the monomer and dimer and would elute
preferentially with 400 mM imidazole. Adopting this refined
purification protocol; we found that, following a 2-ml wash with 250 mM imidazole, the eluted protein was most concentrated in
the first 1-ml elution with 400 mM imidazole. Although the
trimer was clearly apparent in this elution fraction (Fig.
2C, lane 2), the monomer and dimers were also at
a higher concentration than in the preceding or subsequent fractions.
As with the dimer, 4 M
-mercaptoethanol dissociated the
trimer, with only the monomer band apparent on SDS-PAGE (data not
shown). To assess the possibility that the oligomerization of EmrA was
in fact SDS-induced aggregation of the protein, we also ran the
EmrA-(1-390) protein on native PAGE (7.5% polyacrylamide gels/Tris-glycine buffer (pH 7.5)), upon which at least three bands
were visible (Fig. 2D), indicating that the oligomerization of EmrA-(1-390) is not an artifact of the SDS treatment. As a further
test for dimerization, EmrA-(1-390) was subjected to gel chromatography, which yielded two protein elution peaks, indicative of
the monomer and the dimer (Fig. 2E). The two proteins
separated by gel chromatography were confirmed as the monomer and dimer by SDS-PAGE (Fig. 2G), and the dimer dissociated in the
presence of 4 M
-mercaptoethanol (Fig. 2G,
lane 6). Consistent with the identity of each protein as
T7-EmrA-(2-390)-His6, both the monomer and dimer
cross-reacted with anti-polyhistidine antibodies (Fig. 2H),
indicating that both proteins had a C-terminal His tag, whereas N-terminal protein sequencing confirmed that both proteins carried an
N-terminal T7 tag. From these experiments we concluded that EmrA-(1-390) forms dimers and trimers.
-helix that anchors
EmrA to the membrane. The finding that EmrA-(1-56) was exclusively
membrane-bound provides further evidence to support our conclusion
(Fig. 2A, lane 7). There is a predominant band
that migrates with an apparent molecular mass of about 12 kDa,
suggestive of an EmrA-(1-56) dimer because the calculated molecular
mass of the monomer is 8.8 kDa. This behavior suggested that
EmrA-(1-56) contains a dimerization domain and provided a plausible
explanation for the fact that the EmrA-(49-390) protein migrates as a
single band (Fig. 2A, lane 2), whereas two bands
are clearly apparent for EmrA-(1-390) (Fig. 2A, lane
5) and EmrA-(15-390) (Fig. 2A, lane 4).
Interestingly, we noted that the sequence for the N-terminal
-helix
that anchors EmrA to the membrane contains a consensus sequence for a
leucine zipper (e.g. counting from Leu23 there
are Leu, Leu, Ala, and Leu residues positioned at intervals of 1, 8, 15, and 22 residues, respectively), which could lead to the formation
of a coiled-coil. To test this hypothesis, we truncated EmrA at
position 29, to produce a protein that would still retain most of the
-helix and therefore would be expected to be membrane-bound but
would lack the first leucine of the zipper motif, weakening the dimer
interactions. Consistent with our hypothesis, the EmrA-(29-390)
protein was monomeric and predominantly membrane-bound, migrating as a
single band to a position similar to that of the smaller
EmrA-(49-390), presumably because EmrA-(29-390) is a membrane protein
(Fig. 2A, lane 2). We conclude that membrane association alone is insufficient to drive dimerization and that the EmrA-(29-390) construct lacks a functional dimerization domain.
-helix, comprising residues 96-213, sandwiched by
-sheet
structure at the N- and C termini; overall the periplasmic domain has a
37.5%
-helix and a 23%
-sheet structure. To test this latter
prediction, we determined the CD spectra for the EmrA-(49-390) protein, which was predicted to have 31%
-helix and 20.1%
-sheet (Fig. 3A).
Considering the possible errors in determining the protein
concentration and their effect upon the CD analysis, this analysis is consistent with the secondary structure prediction from
Jpred. We also analyzed the sequence using the program COILS (25),
which predicted that residues 95-144 and 156-182 could form the two
helices of a coiled-coil. These helices are shorter than the helical
region predicted by Jpred because of the presence of proline
residues at positions 148 and 197, which are excluded from the helices
predicted by COILS. Furthermore, the MULTICOIL (26) program predicted
that these helices would form dimers and, with a higher probability,
trimers. Consistent with this prediction, we found that under native
PAGE conditions, a concentrated sample of EmrA-(49-390)
formed oligomers (data not shown), indicating a propensity of the
periplasmic domain to oligomerize; but presumably these contacts are
less stable than those in EmrA-(1-390) because we do not see oligomers
of EmrA-(49-390) on SDS-PAGE unless the gel was severely overloaded
with the protein. Furthermore, the insertion of cysteine residues at
the N- and C-terminal ends of EmrA-(49-390) stabilized the formation
of dimers and trimers by the periplasmic domain, which were readily
identified by SDS-PAGE.2
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Fig. 3.
CD spectral analysis of EmrA-(49-390) and
HmrA-(48-390). A, the CD spectra for EmrA-(49-390)
predict a secondary structural content of 31% -helix and 20.1%
-sheet, consistent with the Jpred prediction of a periplasmic domain
consisting of an
-helical coiled-coil domain (37.5%) and a
-sheet domain (23%). The CD spectra for EmrA-(49-390) in the
presence of GdmCl indicate a loss of signal at 222 nm, suggesting that
the unfolding of EmrA-(49-390) primarily involves a loss of
-helical content. In contrast, a SELCON analysis indicated that the
-sheet content increased to more than 30% in GdmCl concentrations
above 1.25 M, suggesting that the
-sheet domain is more
stable than the
-helical coiled-coil. As indicated by the comparable
CD spectra, HmrA-(48-390) adopts a structure similar to that of
EmrA-(49-390). B, unfolding transition of EmrA+. The sharp
transition is consistent with the highly cooperative unfolding of the
-helical coiled-coil domain.
-helical coiled-coil
domain and a
-sheet domain, the stability of these domains might
differ under progressively denaturing conditions, such as increasing
GdmCl concentrations. Consistent with this prediction we found that the
CD signal at 222 nm, indicative of the
-helical content
of the protein, underwent a sharp transition (e.g.
midpoint ~ 1.125 M) to a lower level with increasing
concentrations of GdmCl, probably because of the highly cooperative
unfolding of the
-helical coiled-coil (Fig. 3B).
Moreover, analysis of the individual CD traces using the secondary
structure prediction program SELCON indicated, in contrast to the
-structure, a decrease in the
-helical content of the protein
with an increasing GdmCl concentration, consistent with the predicted
two-domain structure.
-barrel attached to a
long
-helical coiled-coil. Analytical ultracentrifugation was used
to analyze the protein shape. First, a sedimentation equilibrium
analysis was used to assess the oligomeric state of EmrA-(49-390).
These measurements indicated that the protein is monomeric, with an
average molecular mass of 41 kDa, which is consistent with the
calculated value of 39.6 kDa. To assess the symmetry of EmrA-(49-390),
we measured the sedimentation coefficient (s
1
protein) of 4.5 × 10
7 cm2
s
1, which compares with a value of 5.5 × 10
7 cm2 s
1 from boundary
spreading sedimentation velocity data, and gave a measure of the
hydrodynamic radius (Rh) of the protein of 4.7 nm, which is highly comparable with the measured
Rs value of 4.3 nm. Assuming EmrA-(49-390) is a
prolate ellipsoid, it is possible to calculate the axial ratio and
estimate the protein dimensions (Table
I). EmrA-(49-390) is predicted to have
dimensions of 27 × 2.3 nm (i.e. 270 × 23 Å) at
20 °C.
Hydrodynamic parameters for the periplasmic domain of EmrA
(EmrA-(49-390))
), were:
s
emrB strain of E. coli (12). Furthermore, the operon encoding VceAB also
encodes the OMP VceC, but VceAB expression is sufficient to complement the deoxycholate sensitivity of a
tolC strain of E. coli (12), indicating that these transporters can operate as bi-
and tripartite systems. Indeed, recent studies have shown that the
MexJK efflux pump of Pseudomonas aeruginosa does not require
the OMP OprM for efflux of the biocide triclosan, but it is required
for efflux of the antibiotics tetracycline and erythromycin (27).
The susceptibility of the acrA mutant N43 expressing EmrAB,
EmrA-(49-390)-EmrB, and HmrAB to various drugs: MIC measurements
for the three N43 transformants
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Fig. 4.
The periplasmic domain of EmrA
possesses a drug-binding site. A, Stern-Volmer plots
for EmrA-(49-390) in the absence (closed circles) of drugs
and in the presence of 39 µM FCCP (open
circles), 19 µM DNP (closed inverted
triangles), 48 µM CCCP (closed squares),
20 µM nalidixic acid (open inverted
triangles), and 53 µM chloramphenicol (open
squares). The difference in the titration curves is indicative of
drug binding, and the lower end points, obtained in the presence of
drugs, indicate a decrease in the exposure of the tryptophan residues
of EmrA-(49-390) upon binding the drug. Note that chloramphenicol,
which is not an Emr substrate, has little effect on the KI titration
curve, consistent with the other drugs binding specifically.
B, Stern-Volmer plots for EmrA-(49-390) (closed
and open circles) and HmrA-(48-390) (closed and
open inverted triangles) in the absence and presence of 39 µM FCCP, respectively. C, Stern-Volmer plots
for HmrA-(48-390) alone (closed circles) and in the
presence of 19 µM nalidixic acid (open
circles) and 17 µM erythromycin (closed
inverted triangles).
acr background. We conclude from
this analysis that EmrA is a drug-binding protein and is likely to play
a role in the direct transfer of drugs from EmrB to TolC.
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Fig. 5.
Titration of the drug binding site of
EmrA. The fluorescence intensity of EmrA-(49-390) (closed
circles) and HmrA-(48-390) (open triangles) was
measured in the presence of the indicated concentrations of FCCP (to
give F0) and after the addition of 1 M KI (to give F). The curve through the
EmrA-(49-390) data points is the best-fit obtained by a nonlinear
regression fit of the data to an equation with hyperbolic and linear
functions, indicating a maximal quench in the fluorescence of 8.1 (±2.8)% and a Kd for the specific
EmrA-(49-390)-FCCP complex of 4.2 (±2.6) µM. For
HmrA-(48-390), F0/F decrease in a
linear manner, paralleling the behavior of EmrA-(49-390) at high FCCP
concentrations, which is indicative of nonspecific binding.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helix, whereas residues
47-390 are arranged into a soluble periplasmic domain. Indeed, we
found that EmrA-(15-390) was membrane-bound, whereas EmrA-(49-390)
was soluble, indicating that residues 15-48, but not 1-15 or 49-390,
are necessary for membrane insertion. Furthermore, EmrA-(29-390) is
partially soluble, suggesting that the membrane-spanning helix starts
between positions 15 and 29. We predict that the periplasmic domain of
EmrA comprises a short
-sheet domain (residues 48-95), a
large
-helical domain that is arranged into a coiled-coil (residues
96-213), and a large
-sheet domain (residues 214-374) with a short
C-terminal helix (residues 375-387). This secondary structure
prediction is reasonably consistent with a CD analysis of
EmrA-(49-390). In common with the MFP AcrA (9) we found that the
periplasmic domain of EmrA is highly elongated, with predicted
dimensions of 27 × 2.3 nm. EmrA is predicted to have a stretch of
110 residues, with an
-helix structure, which is arranged into a
coiled-coil. Assuming that there are 3.5 amino acids/helix turn, with a
pitch of 0.51 nm/turn, we would expect this domain to have dimensions
of 17 × 2 nm. Thus, both our CD and hydrodynamic data are
reasonably consistent with the Jpred secondary structure prediction of
a two-domain protein, e.g. a globular domain with a largely
-sheet structure capping an
-helical coiled-coil domain.
-helical coiled-coil structure, which
is a common motif in oligomeric proteins; the MULTICOIL program
predicts that the coiled-coil domain will form dimers and trimers.
Consistent with this prediction, we found that the soluble periplasmic
domain of EmrA (e.g. EmrA-(49-390)) formed oligomers (at
relatively high protein concentrations), but the dimers formed by
membrane-bound whole EmrA (e.g. EmrA-(1-390)) were more
stable. We note that both membrane-bound and soluble AcrA form dimers
and trimers (31). However, AcrA differs from EmrA in that it
uses a lipid moiety to anchor it to the inner membrane, indicating that
the oligomerization site for AcrA lies within the periplasmic domain.
Another MFP, HlyD, was also shown to form trimers (8), suggesting that
this is a common feature of MFPs. It seems most likely that a leucine
zipper motif that runs through the N-terminal helix of EmrA acts as a
dimerization domain, which stabilizes dimers of EmrA-(1-390) relative
to those of EmrA-(49-390). Perhaps formation of the dimer provides a
scaffold for trimerization, which results from interactions between the periplasmic domains of EmrA. In such a trimer the third leucine zipper
motif would be "free." Thus, it is of interest to note that both
the putative N-terminal (e.g. Leu7,
Ile14, Leu21, Leu28, and
Val35, which span putative helix 1 between residues 13 and
38) and C-terminal helices (e.g. Leu473,
Ile480, Ile487, and Leu494, which
span putative helix 14 between residues 482 and 504) of EmrB contain
leucine zipper motifs, which might interact with the free leucine
zipper of the EmrA trimer to form a stable EmrA-EmrB complex. No
leucine zipper motifs spanning the other putative helices of EmrB are
apparent. Recent studies of HylD indicate that its cytosolic domain
mediates transduction of the substrate binding signal to the
periplasmic domain to trigger recruitment of TolC (32). Thus, it is
inviting to speculate that substrate binding to EmrB triggers
communication between EmrB and EmrA via the leucine zippers, with
signal propagation to the periplasmic domain of EmrA.
/
-barrel that forms trimers (33). This
is suggestive of a related structure and function for these proteins,
possibly with both acting to channel drugs across the periplasm.
Indeed, the trimeric structure of EmrA is suggestive of the formation
of a six-helix barrel, which could form a connecting channel with TolC.
However, both TolC and EmrA are predicted to be sufficiently long to
span most, if not all, of the periplasmic space, bringing into question
how this interaction might be achieved if only the ends of the helical
channels are involved. An alternative hypothesis might be one in which
each
-helical coiled-coil of trimeric EmrA would act like "arms to grab" TolC, inducing the periplasmic end of TolC to adopt an open confirmation. Consistent with this hypothesis, recent studies indicate
that a ring of aspartate residues (34) and an intramolecular salt
bridge (35) at the periplasmic end of TolC control the opening of the
tunnel; their interaction with the MFP could be used to control the
opening of TolC. However, there is evidence that the IMP and MFP, which
are coupled, can work independently of the OMP (12, 27). It is possible
that the MFP performs a role similar to that of TolC but channels the
drugs to the outer membrane. Thermodynamically, the delivery of
hydrophobic drugs to the outer membrane would be favorable.
-helix, AcrA is
anchored to the membrane by a lipid moiety. It has also been suggested that drugs can bind to the headpiece of AcrB and be channeled into the
central pore region (5). The headpiece contains vestibules that are
open to the periplasm, which could be used to channel drugs to the pore
at the center of the headpiece. Drugs delivered to the pore from the
periplasm could then be delivered to TolC. Recent studies have shown
that swapping the periplasmic domains of the two RND antiporters, AcrB
and AcrD, which differ in their drug specificity, results in a change
in the drug specificity of the chimeric proteins (36), thus providing
evidence that the periplasmic domains of AcrB are involved directly in
drug binding. Herein we provide evidence that EmrA binds transported drugs, and it is tempting to speculate that EmrA may serve a similar role to that of the periplasmic domains of RND antiporters in drug
binding and transfer to the OMP.
-sheet domain of EmrA above EmrB at the
surface of the membrane, with the
-helices radiating out across the
periplasm in position to contact TolC when triggered by the binding of
drugs to the
-sheet domain of EmrA. The
-helical coiled-coils of
EmrA would grab TolC so as to position the
-helical barrel of TolC
above the
-sheet domain of EmrA. The drug could then be released
from the
-sheet domain of EmrA, allowing it to diffuse into the
channel formed by the TolC trimer. Although one can envisage the drug
moving from a less hydrophobic site on EmrA/EmrB to a more hydrophobic
site on TolC, it is puzzling as to how TolC rids itself of the drug. Clearly a detailed understanding of the function of EmrA will require
knowledge of the three-dimensional structure, and toward this end we
have recently succeeded in crystallizing EmrA.
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FOOTNOTES |
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* This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC) and Wellcome Trust (to A. R. W.). The CD facility at the University of Glasgow and the National Center for Macromolecular Hydrodynamics at the University of Nottingham are both supported by the BBSRC and the Engineering and Physical Sciences Research Council (EPSRC).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: Centre for
Infectious Diseases, Wolfson Research Institute, University of Durham, Queen's Campus, Stockton-on-Tees TS17 6BH, United Kingdom. Tel.: 44-1642-333836; Fax: 44-1642-333817; E-mail:
a.r.walmsley@durham.ac.uk.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M209457200
2 M. I. Borges-Walmsley and A. R. Walmsley, unpublished data.
3 The fluorescence changes associated with the specific binding of nalidixic acid were too small to be distinguished reliably from those associated with nonspecific binding.
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ABBREVIATIONS |
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The abbreviations used are:
IM, inner membrane;
MFP, membrane fusion protein;
IMP, inner membrane protein (a membrane transporter);
CB, carbenicillin;
MES, 4-morpholineethanesulfonic acid;
MIC, minimum inhibitory concentration;
MOPS, 4-morpholinepropanesulfonic acid;
NTA, nitrilotriacetic acid;
OM, outer
membrane;
OMP, outer membrane protein (an /
-barrel protein channel);
RND, resistance-nodulation-cell division family of membrane
transporters;
FCCP, carbonyl cyanide
p-trifluoromethoxyphenyl-hydrazone;
CCCP, carbonyl cyanide
m-chlorophenyl-hydrazone;
DNP, 2,4-dinitrophenol.
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