Download Molecular Basis of Multidrug Transport by ATP

J. Mol. Microbiol. Biotechnol. (2001) 3(2): 185-192.
Symposium
Molecular MechanismJMMB
of ABC Transporters
185
Molecular Basis of Multidrug Transport
by ATP-Binding Cassette Transporters:
A Proposed Two-Cylinder Engine Model
Hendrik W. van Veen1*, Christopher F. Higgins2,
and Wil N. Konings3
1Department
of Pharmacology, University of Cambridge,
Tennis Court Road, Cambridge CB2 1QJ, UK
2MRC Clinical Sciences Centre, Imperial College School
of Medicine, Hammersmith Hospital Campus, Du Cane
Road, London W12 0NN, UK
3Department of Microbiology, Groningen Biomolecular
Sciences and Biotechnology Institute, University of
Groningen, Kerklaan 30, NL-9751 NN Haren, The
Netherlands
Abstract
ATP-binding cassette multidrug transporters are
probably present in all living cells, and are able to
export a variety of structurally unrelated compounds
at the expense of ATP hydrolysis. The elevated
expression of these proteins in multidrug resistant
cells interferes with the drug-based control of cancers
and infectious pathogenic microorganisms. Multidrug
transporters interact directly with the drug substrates.
Insights into the structural elements in drug molecules
and transport proteins that are required for this
interaction are now beginning to emerge. However,
much remains to be learned about the nature and
number of drug binding sites in the transporters, and
the mechanism(s) by which ATP hydrolysis is coupled
to changes in affinity and/or accessibility of drug
binding sites. This review summarizes recent advances
in answering these questions for the human multidrug
resistance P-glycoprotein and its prokaryotic homolog
LmrA. The relevance of these findings for other ATPbinding cassette transporters will be discussed.
Introduction
In the past decade, the importance of multidrug transport
proteins in the simultaneous resistance of cells to multiple
cytotoxic drugs has been recognized increasingly.
Resistance of human cells to anticancer drugs is commonly
associated with high expression of the multidrug resistance
P-glycoprotein (Gottesman et al., 1995) or the multidrug
resistance-associated protein (MRP1) (Hipfner et al., 1999).
These transporters belong to the family of ATP-binding
cassette (ABC) proteins (Higgins, 1992; Holland and Blight,
1999), and pump drugs out of the cell at the expense of
*For correspondence. Email [email protected]; Fax. +44-1223-334040.
© 2001 Horizon Scientific Press
ATP hydrolysis. ABC multidrug transporters are also
expressed in microorganisms in which they confer
resistance to antibiotics and other cytotoxic compounds.
Examples in microorganisms include LmrA in Lactococcus
lactis (van Veen et al., 1998; Putman et al., 2000), Pdr5p,
Ycf1p and Yor1p in Saccharomyces cerevisiae, and Cdr1p
in Candida albicans (Prasad et al., 1995; Taglicht and
Michaelis, 1998; Bauer et al., 2000).
Most eukaryotic multidrug ABC transporters (e.g., Pglycoprotein) comprise four domains which are fused into
a single polypeptide. Two of these domains, the
transmembrane domains, are hydrophobic and span the
membrane multiple times. There is now compelling
evidence that each membrane domain of P-glycoprotein
consists of six transmembrane segments (putative αhelices), a total of twelve transmembrane segments per
P-glycoprotein molecule (Kast et al., 1996; Loo and Clarke,
1999). These domains are thought to form the pathway
through which drug molecules cross the membrane. The
other two domains are hydrophilic nucleotide binding
domains which couple the energy of ATP hydrolysis to the
transport process (Higgins, 1992). There is evidence for
P-glycoprotein that this monomeric, four-domain protein
is the functional unit (Higgins et al., 1997; Rosenberg et
al., 1997; Loo and Clarke, 1999). The protein appears to
have arisen by a gene duplication event, fusing two related
half-molecules, each consisting of one nucleotide binding
domain and one transmembrane domain. In contrast,
lactococcal LmrA appears to be a half-transporter
consisting of an N-terminal transmembrane domain with
six membrane-spanning segments, fused to the nucleotide
binding domain (van Veen et al. , 1996). LmrA is
homologous to each of the two halves of P-glycoprotein,
and self-associates into a homodimer to form a full
transporter with four core domains (van Veen et al., 2000).
In addition to the four core domains, the eukaryotic MRP
proteins (e.g., MRP1, Ycf1p and Yor1p) have an N-terminal
membrane domain with 5 membrane spanning segments
(Tusnády et al., 1997).
ABC transporters share significant sequence similarity,
and may have a similar 3D-structure and mechanism.
Hence, studies on any one member of the ABC family, be
it of eukaryotic or prokaryotic origin, may provide a template
for all other members. Various approaches such as
mutational analyses, and biochemical and pharmacological
characterizations have yielded a wealth of information
about structure-function relationships in ABC multidrug
transporters and their drug substrates. However, there is
still a considerable controversy about the mechanisms by
which these proteins pump drugs from the interior of the
cell to the external environment. In this review we set out
to highlight key recent advances in our understanding of
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186 van Veen et al.
the molecular basis of drug recognition and translocation
by ABC multidrug transporters, in particular LmrA and Pglycoprotein, and discuss some implications of these
findings for the mechanism of other ABC transporters.
Drug Specificity and Transport Models
Insight into the molecular basis of the drug specificity of
multidrug transporters is crucial for the development of new
drugs which are not transported, and for the development
of modulators which may inhibit these proteins through
high-affinity binding. Unlike other transporters in the ABC
family, each of which is relatively specific for its substrate
(e.g., amino acid or sugar), multidrug transporters have
an exceptionally broad specificity for structurally dissimilar
compounds. For example, the human P-glycoprotein
exhibits specificity for neutral or cationic cytotoxic drugs,
such
as
anthracyclines,
vinca-alkaloids,
epipodophyllotoxins, taxol, colchicine and actinomycine D.
In addition, the protein is able to interact with compounds
such as steroid hormones, cyclic and linear peptides, lipids,
immunosuppressive agents, and calcium channel blockers
(Ueda et al., 1997). This broad specificity is not only
confined to P-glycoprotein. A similar range of compounds
also interacts with lactococcal LmrA (van Veen et al., 1998),
yeast Pdr5p (Kolaczkowski et al., 1996; Bauer et al., 2000)
and other ABC multidrug transporters. LmrA can even
substitute for P-glycoprotein in human lung fibroblast cells,
suggesting that this type of multidrug resistance efflux pump
is conserved from bacteria to man (van Veen et al., 1998).
Although the drugs which interact with P-glycoprotein
and LmrA have little in common, they do share an
amphipatic structure which allows partitioning of the
molecules into the phospholipid bilayer, and reach their
intracellular targets by passive diffusion through the bilayer
(Frezard and Garnier-Suillerot, 1991; Speelmans et al.,
1996; Ueda et al., 1997). Two models that could account
for the broad specificity propose that multidrug transporters
bind the amphipatic drugs within the cytoplasmic (inner)
leaflet of the membrane, and transport their polar group
across the membrane directly into the external water phase
(vacuum cleaner model) (Raviv et al., 1990; Bolhuis et al.,
1996a), or into the exoplasmic (outer) leaflet of the
membrane from which they would diffuse into the external
water phase (flippase model) (Higgins and Gottesman,
1992). Hence, metabolic energy would be required by LmrA
and P-glycoprotein to catalyze drug transport against the
lipid/water partition coefficient.
If these transporters operate by these mechanisms,
the rate at which the drug would flip back by passive
diffusion from the exoplasmic leaflet to the cytoplasmic
leaflet should be low. There is evidence which supports
this notion, e.g., anthracyclines diffuse from one leaflet to
the other with a half life in order of minutes at physiological
pH (Frezard and Garnier-Suillerot, 1991). The notion that
P-glycoprotein and LmrA are able to transport drugs from
the membrane is supported by the observations that (i)
the lipophilicity and partition coefficient of a drug are key
factors which determine the efficiency of transport by Pglycoprotein (Chiba et al., 1996), (ii) acetoxymethyl esters
of several fluorescent probes accumulate less in Pglycoprotein or LmrA-expressing cells, despite the fact that
the ester moieties are rapidly cleaved by intracellular
esterases and the resulting carboxylates are not substrates
for P-glycoprotein and LmrA (Homolya et al., 1993; Bolhuis
et al., 1996a), and (iii) the kinetics of ATP-dependent
transport of Hoechst 33342 by P-glycoprotein in insideout membrane vesicles is consistent with the transport of
Hoechst 33342 from the lipid phase (Shapiro et al., 1997).
Subsequent kinetic studies on the LmrA-mediated transport
of TMA-DPH (Bolhuis et al., 1996a), and the P-glycoproteinmediated transport of Hoechst 33342 (Shapiro and Ling,
1997a) and LDS-751 (Shapiro and Ling, 1998) revealed a
dependence of the drug extrusion rate on the drug
concentration in the cytoplasmic leaflet of the phospholipid
bilayer, rather than the extracellular leaflet.
Extrusion from the cytoplasmic leaflet of the membrane
could, in principle, explain the broad drug specificity of
multidrug transporters. The ability of drugs to intercalate
into the cytoplasmic leaflet of the membrane would preselect drugs to be transported from other cellular
compounds that are not to be transported. Subsequently,
the interaction between the pre-selected drug and the
transporter would be another important determinant of
specificity. Pharmacological and genetic analyses of the
drug specificity of P-glycoprotein demonstrate that,
although P-glycoprotein transports an exceptionally broad
spectrum of drugs, the transporter does exhibit drug
specificity. This drug specificity is strongly dependent on
the primary structure of the protein (Gottesman et al., 1995).
The presence of specific drug binding site(s) in the Pglycoprotein and LmrA would enable the transport of drug
molecules that are highly concentrated in the phospholipid
bilayer despite being present at low concentrations in the
extracellular and cytoplasmic water phase.
Although drug transport from the cytoplasmic leaflet
of the membrane may be a general mechanism of multidrug
transporters with specificity for amphipatic drugs, including
transporters which are dependent on the proton motive
force rather than ATP hydrolysis such as lactococcal LmrP
(Bolhuis et al., 1996b), other mechanisms may also exist.
In addition to the ability to transport drugs that partition
into the membrane, MRP1 and related multidrug
transporters have specificity for strictly hydrophilic drugs,
e.g. anionic carboxyfluorescein derivatives and certain
glutathione conjugates, which do not partition into the
membrane (König et al. , 1999). Most likely, these
hydrophilic drugs gain access to the transporter from the
aqueous phase, suggesting that two pathways exist for
the entrance of drugs into MRP1 and related transporters,
one from the membrane and the other from the aqueous
phase, or that, alternatively, a single pathway is accessible
from both the aqueous phase and the membrane (Higgins
and Gottesman, 1992).
Determinants of Drug-Protein Interactions
Although multidrug transporters are relatively non-selective,
accumulating experimental evidence supports the
contention that the proteins interact directly with the drugs
through specific drug-protein interactions: (i) multidrug
transporters can be covalently labeled with photo-reactive
drug analogs, and the binding of the drug analogs, prior to
the cross-linking reaction, can be outcompeted by the
Molecular Mechanism of ABC Transporters 187
binding of other drugs (Bruggeman et al. 1992;
Greenberger, 1993; Dey et al., 1997), (ii) small structural
changes in drug molecules may have a great effect on
transport or binding affinities (Klopman et al., 1997; Pajeva
and Wiese, 1998; Ecker et al., 1999; Orlowski and Garrigos,
1999), and (iii) single amino acid substitutions in multidrug
transport proteins can dramatically influence the specificity
for specific drugs (Gottesman et al., 1995; Ueda et al.,
1997).
Structural Elements Required in Drug Molecules
In addition to the bias for lipophilicity, structural elements
in drug molecules appear to influence the recognition by
multidrug transporters (Seelig, 1998; Ecker et al., 1999).
For P-glycoprotein substrates, the structural elements
seem to consist of electron donor units formed by electron
donor (hydrogen bond acceptor) groups which are arranged
in distinct spatial patterns: two electron donor groups with
a spatial separation of 2.5 ± 0.3 Å (Type I unit), and two
electron donor groups with a spatial separation of 4.6 ±
0.6 Å, or three electron donor groups with a spatial
separation of the outer two groups of 4.6 ± 0.6 Å (Type II
unit) (Figure 1). Different types of electron donor groups
can be present in the Type I and Type II units, and the
frequency of their appearance in these units decreases in
the order: >C=O [oxygen in the carbonyl groups of
aldehydes, ketones, acids, esters and amines]; -OR
[oxygen in alkoxy groups]; -NR 3 [nitrogen in quatenary
ammonium groups]; -N= [nitrogen in imines, pyrroles and
pyridines]; RX [halides, X= Cl or F]; -SR [sulfides],
>C(C6H5)2, [ electron orbital of phenyl groups, especially
in diphenyl methylene]; -OH [oxygen in hydroxy groups];NR2 [nitrogen in tertiary amines]; -NHR [nitrogen in
Figure 1. Backbone structures of selected P-glycoprotein substrates. A.
Type I units contain two electron donor groups (designated by “A”)with a
spatial separation of 2.3 Å (see dashed lines). B. Type II units contain either
three electron donor groups with a spatial separation of the outer two electron
donor groups of 4.6 Å, or two electron donor groups with a spatial separation
of 4.6 Å (adapted from Seelig, 1998).
secondary amines]; -NH 2 [nitrogen in primary amines]. Pglycoprotein substrates appear to contain at least one Type
I unit or one Type II unit and the binding of the substrates
to P-glycoprotein increases with the strength and number
of these units. MRP1 substrates share chemical properties
with P-glycoprotein substrates, and often contain at least
one electrically neutral Type I unit together with one
negatively charged Type I unit or two electrically neutral
Type I units (Seelig et al., 2000). The negatively charged
Type I unit includes the carboxylic group (e.g., in glutathione
conjugates), the sulfonic acid group (e.g., taurocholate),
the sulfate group (e.g., sulfate conjugates), the cyclic
phosphodiester group (e.g., bucladesine), or the
mesomeric nitro group (e.g., 2,4-dinitrophenyl glutathione).
Compounds with cationic Type I units, which are good
substrates for P-glycoprotein, are not transported by MRP1.
Structural Elements Required in Multidrug Transport
Proteins
Our understanding of the structural elements in multidrug
transport proteins which dictate drug specificity is still poor
and should come from structural analysis of transporters
and transporter-drug complexes. Membrane proteins are
notoriously refractory to structural analysis by 2D- and 3Dcrystallization techniques. Recently, crystal structures at
2.7 Å resolution in the absence and presence of drug were
obtained for a soluble transcription regulator BmrR from
Bacillus subtilis (Zheleznova et al., 1999). BmrR activates
the expression of the secondary multidrug transporter Bmr
in response to the binding of amphipatic compounds.
Tetraphenylphosphonium appears to penetrate into the
hydrophobic core of BmrR, where it forms hydrogen bonds
and stacking interactions with hydrophobic and aromatic
residues, and makes an ion pair interaction with a buried
glutamic residue Glu134 (Zheleznova et al., 1999; VázquezLaslop et al., 1999). Since tetraphenylphosphonium is also
transported by the Bmr transporter (Neyfakh et al., 1991),
similar drug-protein interactions may occur in Bmr and other
multidrug transporters (Zheleznova et al., 2000). Indeed,
the presence of acidic residues within transmembrane
segments of secondary multidrug transporters (e.g., MdfA
and EmrE in Escherichia coli) (Edgar and Bibi, 1999; Muth
and Schuldiner, 2000; Yerushalmi and Schuldiner, 2000)
and QacA in Staphylococcus aureus (Paulsen et al., 1996))
was shown to be been related to the cation specifity of
these transporters.
Although acidic residues in transmembrane segments
may also play a role in the cation selectivity (or positively
charged residues in the anion selectivity) of ABC multidrug
transporters, P-glycoprotein, LmrA and others do not
possess acidic residues in their membrane domains, and
yet, do transport cationic amphipatic drugs. Hence,
alternative mechanism(s) for cation selectivity must exist.
Interestingly, it has been shown that cations can bind to
the face of the aromatic ring structures of tyrosine,
phenylalanine and tryptophan residues (Dougherty, 1996).
Since, in the hydrophobic environment of the phospholipid
bilayer this binding can be as strong as the electrostatic
interactions between ion pairs, aromatic residues may
determine cation selectivity in both ABC multidrug
transporters and secondary multidrug transporters. This
notion is supported by the observation that site-directed
188 van Veen et al.
substitution of aromatic residues in P-glycoprotein does
affect drug specificity (Ueda et al., 1997; Kwan et al., 2000).
Analysis of the transmembrane α-helices in P-glycoprotein
and LmrA has revealed that aromatic residues and polar
amino acid residues with hydrogen donor side chains are
often clustered together on one side of a helix, with amino
acid residues with non-hydrogen bonding side chains on
the other side (data not shown, and Seelig et al., 2000).
Hence, the transmembrane segments could be oriented
with their non-interactive residues facing the hydrophobic
phospholipid bilayer, and their interactive residues facing
a translocation pore. Within this pore, electrons may
enable cation binding and may even provide a “slide guide”
system for cationic drugs, whereas hydrogen bonds and
stacking interactions facilitate the interaction with
electroneutral moieties within the amphipatic drug
molecules. In the case of MRP1, the hydrogen bonding
face within transmembrane segments also contains cationic
residues which may determine anion selectivity of the
transporter (Seelig et al., 2000).
Drug Binding Sites
Evidence has been obtained that the drug-protein
interactions which determine the binding specificity of
multidrug transporters are organized within specific drug
binding sites or domains in the proteins. Independent
photoaffinity labeling studies of P-glycoprotein involving the
1,4-dihydropyridine derivatives azidopine (Bruggeman et
al., 1992), iodoaryl-azidoprazosin (Greenberger, 1993) and
iodoaryl-azidoforskolin (Busche et al., 1989), and the
daunomycin derivative iodomycine (Demmer et al., 1997)
have identied the same two photobinding regions within
the transmembrane domains, encompassing
transmembrane segments 5 and 6 in the N-terminal half
and transmembrane segment 11 and 12 in the C-terminal
half. The inhibition of photoaffinity labeling in these regions
by many structurally unrelated substrates of P-glycoprotein
suggest that the photobinding regions contribute to a single
binding site that interacts with many different drugs
(Bruggeman et al., 1992) or that, alternatively, each
photobinding region may be part of a separate site, giving
two distinct drug binding sites per P-glycoprotein monomer
(Dey et al., 1997).
A number of observations is consistent with the notion
that P-glycoprotein possesses at least two separate drug
binding sites. First, the kinetic analysis of competitive, noncompetitive and cooperative interactions between different
transport substrates implied the presence of two transportcompetent drug binding sites in P-glycoprotein (Ayesh et
al., 1996). Second, rhodamine 123 and Hoechst 33342
(Shapiro and Ling, 1997b), and colchicine and synthetic
hydrophobic peptides (Sharom et al., 1996) stimulated the
P-glycoprotein-mediated transport of each other. Shapiro
and Ling suggested that this effect may arise from positively
cooperative interactions between two transport-competent
drug binding sites, denoted the R site and H site. Third, a
biphasic fluorescence quenching by vinblastine and
verapamil of 2-[4'-maleimidyl-anilino]naphthalene 6sulfonic acid (MIANS)-labeled P-glycoprotein was
observed, suggesting a two-affinity binding of these drugs
to P-glycoprotein (Sharom et al., 1999). Biphasic binding
of vinblastine and verapamil to P-glycoprotein was also
observed using a radioligand binding assay
(Döppenschmitt et al., 1999). Fourth, the modulator cis(Z)flupentixol increased the affinity of iodoarylazidoprazosin
for the C-terminal half of P-glycoprotein (C site) without
changing the affinity for the N-terminal half (N site) (Dey et
al., 1997). In addition, iodoarylprasozin binding to these
sites was differentially inhibited by both vinblastine and
cyclosporin. Collectively, these data suggest the presence
of at least two drug binding sites in P-glycoprotein with
overlapping drug specificities but with non-identical drug
binding affinities.
ATP-Dependent Drug Translocation by P-Glycoprotein
P-glycoprotein requires ATP hydrolysis for activity. Several
observations provide strong support to an alternating
catalytic sites model for ATP hydrolysis in which the ABC
domains of P-glycoprotein act alternately to hydrolyse ATP:
(i) mutations (Urbatsch et al. , 1998) or covalent
modifications (Senior and Bhagat, 1998; Loo and Clarke,
1999) that inactivate ATP hydrolysis by one of the ABC
domains in P-glycoprotein abolishes all drug-stimulated
ATPase activity, demonstrating catalytic cooperativity
between the two ABC domains in the transporters, (ii) orthovanadate, a potent inhibitor of P-glycoprotein and LmrAassociated ATPase and drug transport activities, permits
only a single turnover of P-glycoprotein by trapping either
the first or the second ABC domain in a catalytic transition
state conformation, thus preventing the other ABC domain
from doing so (Urbatsch et al., 1995), (iii) photo-affinity
labeling experiments with azido-nucleotides suggest that
ATP binding to one ABC domain stimulates ATP hydrolysis
at the other ABC domain (Ueda et al., 1999), and that 1
mole of Mg-8-azido-ADP is bound per mole of Pglycoprotein after hydrolysis of Mg-8-azido-ATP (Senior et
al., 1995).
In order to mediate drug transport, P-glycoprotein must
couple ATP hydrolysis to conformational changes which
alter drug binding affinity and/or accessibility of drug binding
sites. Indeed, photoaffinity labeling of vanadate-trapped
P-glycoprotein provided evidence that the protein exhibits
a reduced affinity for drugs upon the hydrolysis of ATP
(Ramachandra et al., 1998). In addition, conformational
changes in P-glycoprotein upon binding and/or hydrolysis
of ATP, and upon binding of drugs, have been detected by
Fourier transform infrared spectroscopy (Sonveaux et al.,
1996), cross-linking of cysteine residues introduced in the
protein (Loo and Clarke, 1999), immunoreactivity of the
protein with mAb UIC2 (Mechetner et al., 1997), partial
trypsin digestion (Wang et al., 1998), intrinsic tryptophan
fluorescence measurements (Sonveaux et al., 1996, 1999),
and fluorescence labeling of the ABC domains (Sharom et
al. , 1999), indicating the existence of different Pglycoprotein conformations associated with different steps
in the transport process. Secondary and tertiary structure
changes induced by nucleotide binding and hydrolysis have
also been detected in LmrA (Vigano et al., 2000).
Two models have been proposed to explain the
conformational coupling between the ABC domains and
drug binding sites in P-glycoprotein. The first one, the
alternating access/single-site model (Tanford, 1983;
Molecular Mechanism of ABC Transporters 189
Figure 2. Drug translocation models proposed for P-glycoprotein. A.
Alternating access/single-site model. The two membrane domains of Pglycoprotein may form a single drug binding site which alternates between
high- and low-affinity conformations exposed at the inner and outer
membrane surface, respectively, during the transport process. B. Fixed
two-site model. In this model, the N- and C-terminal membrane domains in
P-glycoprotein form distinct drug binding sites, an “on-site” and an “of fsite”, which are part of the translocation pathway. C. Alternating access/
two-site models. P-glycoprotein contains two drug binding sites, each with
alternate access from the two sides of the membrane, which alter their
accessibility simultaneously (1), or in a sequential (2) or alternating (3)
fashion. Out and in refer to the outside and inside of the membrane,
respectively.
Bruggeman et al., 1992), proposes the presence of a single
drug binding site in the protein which is alternately exposed
to the inner or the outer surface of the phospholipid bilayer,
with a concomitant change in drug binding affinity (Figure
2A). In this model, the observation of two drug binding sites
in P-glycoprotein reflects the presence of two different
protein conformations within the population of transporter
molecules: one population of molecules with a high affinity
binding site at the inner membrane surface, and a second
population of molecules with a low affinity binding site at
the outer membrane surface. ATP hydrolysis is required to
interconvert one population into the other, yielding a ratio
of 2 molecules of ATP hydrolyzed per drug molecule
transported. This ratio is consistent with the experimentally
determined coupling ratio which ranges between 1 and 3
(Eytan et al., 1996; Ambudkar et al., 1997; Shapiro and
Ling, 1998). However, a major drawback of the single-site
model is its inconsistency with the observed cooperative
and non-competitive interactions between transport
substrates, and the reciprocal stimulation of the Pglycoprotein-mediated transport of one drug by another
drug.
The second model, the fixed two-site model, proposes
the presence of two drug interaction sites: an “on-site” with
high drug binding affinity in the C-terminal half of the protein,
and an “off-site” with low affinity in the N-terminal half (Dey
et al., 1997) (Figure 2B). This interesting model suggests
a potential link between the mechanism of ABC transporters
and the mechanism of ABC channels (e.g., see Gadsby
and Nairn, 1999, for a review on the gating mechanism of
the cystic fibrosis transmembrane conductance regulator
(CFTR)). Upon ATP hydrolysis at one ABC domain, a
conformational change decreases the affinity of the onsite for drugs, and as a result, the drug moves from the onsite to the off-site through a pore-like structure.
Subsequently, ATP-hydrolysis at the second ABC domain
drives drug translocation from the off-site to the external
medium. It has also been proposed that drug translocation
from the off-site may occur without an additional
requirement for ATP hydrolysis (Ambudkar et al., 1999).
This suggestion reduces the fixed two-site mechanism to
an alternating access/single-site mechanism combined with
a “slide guide” system involving a static low-af finity drug
binding site. Since both models assume the presence of a
single on-site, they have similar drawbacks as the singlesite model mentioned above.
Besides single-site models, models can be proposed
which are based on alternating access/two-site
mechanisms (Figure 2C). A key feature of these
mechanisms is the requirement of drug binding to two
separate sites with overlapping specificities at the inner
membrane surface of P-glycoprotein. The hydrolysis of ATP
results is the movement or altered accessibility of both sites
from the inner surface to the outer surface, simultaneously
or in a sequential or alternating fashion, with a concomitant
change to low affinity. At the outer membrane surface, both
drug molecules dissociate from the transporter. In contrast
to the single-site mechanisms, the two-site mechanisms
are consistent with cooperative and non-competitive
interactions between transport substrates, and the
reciprocal stimulation of the P-glycoprotein-mediated
transport of drugs by other drugs.
Drug Transport Cycle of LmrA
Recently, the mechanism of drug transport by the
lactoccocal LmrA transporter has been studied, and strong
evidence has been obtained that supports an alternating
two-site (two-cylinder engine) mechanism and that
excludes the single-site mechanisms proposed for Pglycoprotein (van Veen et al., 2000). First, a stoichiometry
of two molecules of vinblastine bound per transporter
molecule of LmrA was determined. Second, vinblastine
equilibrium binding experiments demonstrate the presence
of a low-affinity vinblastine binding site in the LmrA
transporter on the outside surface of the membrane, which
is allosterically coupled to a high-affinity vinblastine binding
site in the protein on the inside surface of the membrane.
Only the low-affinity, outside-facing vinblastine binding site
is accessible in the ADP/vanadate-trapped LmrA
transporter, providing evidence for the coupling between
ATP hydrolysis and the outwardly-directed transbilayer
movement of the high-affinity vinblastine binding site with
a concomitant change to low affinity. Third, competitive
190 van Veen et al.
inhibitors of vinblastine binding also showed behaviour
consistent with two drug binding sites. Finally, these two
sites were shown to be directly related to drug transport
by the observation of reciprocal stimulation of LmrAmediated vinblastine and Hoechst 33342 transport at low
drug concentrations, and reciprocal inhibition at high drug
concentrations.
In this alternating two-site (two-cylinder engine)
mechanism the hydrolysis of ATP by the ABC domain of
one half of the transporter is coupled to drug efflux through
the movement of the inside-facing, high-affinity drug binding
site [which binds a drug molecule in the inner leaflet of the
membrane] to the outside of the membrane with a
concomitant change to low affinity [resulting in the release
of the drug molecule into the extracellular medium] ( Arrow
1 and 2 in Figure 3). In the ADP/vanadate-trapped
transporter, the high-affinity drug binding site is occluded.
In addition, ATP binding and/or hydrolysis must facilitate
the return of an unliganded outside-facing low-affinity site
to an inside-facing high-affinity site. Presumably, ATPbinding by the ABC domain of the other half molecule is
also induced. The alternating two-site model for drug
binding and transport proposed for LmrA is consistent with
the alternating catalytic sites mechanism proposed for ATP
hydrolysis by P-glycoprotein (Senior and Gadsby, 1997),
and provides a model which links the drug binding and
catalytic cycles.
Two-Cylinder Engine: A General Mechanism for ABC
Transporters?
The alternating two-site (two-cylinder engine) mechanism
may also apply to other ABC transporters. First, the
mechanism may be relevant for P-glycoprotein. LmrA and
P-glycoprotein are homologous proteins with overlapping
drug and modulator specificities and a similar domain
organization (van Veen et al., 1998, 2000). The current
data obtained for LmrA are inconsistent with the singlesite models proposed for P-glycoprotein. In contrast, most
data reported for P-glycoprotein could be consistent with
various two-site mechanisms, including the two-cylinder
engine model proposed for LmrA. Second, the two-cylinder
engine mechanism may be relevant for mammalian MRP
proteins, which play an important role in (i) detoxification
by mediating the excretion of conjugation products of drug
metabolism into bile and urine, and (ii) defense against
oxidative stress by influencing the GSH/GSSG ratio
(Hipfner et al., 1999; König et al., 1999). Hence, these drug
pumps are able to efflux glutathione, glucoronide and
sulfate conjugates, GSSG, as well as non-conjugated drugs
in cotransport with reduced glutathione (Cole and Deeley,
1998). The stimulation of the transport of non-conjugated
drugs by glutathione and vice versa (Loe et al., 2000a,b;
Evers et al., 2000) seems incompatible with single-site
mechanisms, and may indicate the presence of two
positively cooperative transport-competent substrate
binding sites in the protein, one for glutatione and the other
for unconjugated drugs (Evers et al., 2000). The recent
observation that the ability of MRP1 to transport
anthracyclines and 17ß-estradiol 17(ß-D-glucuronide) is
linked to the second half-transporter in the protein (Stride
et al., 1999), is consistent with this notion. Third, the two-
Figure 3. Alternating two-site (two-cylinder engine) model proposed for LmrA.
This model is related to the model depicted in Figure 2C(3). Rectangles
represent the transmembrane domains of LmrA. Circles, squares and
hexagons represent different conformations of the nucleotide-binding
domains. The ATP-bound (circle) state is associated with a high-affinity
drug binding site on the inside of the transporter. The ADP-bound (square)
state is associated with a low-affinity drug binding site on the outside of the
transporter. The ADP-Pi (hexagonal) state is associated with an occluded
drug binding site, and represents the ADP/vanadate-trapped form of the
ABC domain. In and out refer the inside and outside of the phospholipid
bilayer, respectively. In the presence of ATP, the transport cycle turns counter
clockwise (as indicated by Arrow 1 and 2). Arrow 1, ATP hydrolysis at the
second ABC domain in the LmrA dimer is coupled to (i) drug efflux at the
second membrane domain through the movement of a liganded insidefacing high affinity site to the outside of the membrane with the concomitant
change to low-affinity, via a catalytic transition intermediate in which the
transport site at the second membrane domain is inaccessible, (ii) the
reorientation of an empty outside-facing low-affinity site at the first membrane
domain to an inside-facing high-affinity site, and (iii) ATP binding at the first
ABC domain. Arrow 2, the first and second LmrA molecule in the dimer
have reversed their relationship, and the next ATP hydrolysis step will occur
at the first ABC domain (adapted from van Veen et al., 2000).
cylinder engine mechanism may also be relevant for
prokaryotic binding protein-dependent ABC transporters
involved in the uptake of solutes (e.g., amino acids and
sugars). In general, these transporters contain four core
domains in the translocator, analogous to the domains of
P-glycoprotein, and an additional extracellular solutebinding protein. Surprisingly, the osmoregulated ABC
transporter OpuA in L. lactis possesses two extracellular
solute-binding proteins of which one is covalently linked to
the N-terminal membrane domain and the other to the Cterminal domain (van der Heide and Poolman, 2000). The
stoichiometry of two binding proteins per translocator
complex raises questions about the observations for the
bacterial periplasmic histidine, ribose and maltose
permeases, each of which makes use of a soluble
periplasmic solute-binding protein, that only a single binding
protein interacts with the dimeric membrane complex and
that each of the two lobes of a single binding protein interact
with a separate membrane domain in these transporters
(Liu et al., 1998; Ehrmann et al., 1998; Park et al., 1999).
However, if OpuA would operate by an alternating two-site
mechanism, each of the two binding-proteins would interact
with the translocator compex in one half of the catalytic
Molecular Mechanism of ABC Transporters 191
cycle, giving two interactions per cycle, and this would be
consistent with the available data for the periplasmic
permeases.
Much remains to be learned about the molecular
mechanism(s) of ABC transporters. At the present it is
unknown if ABC transporters operate by a common
mechanism, or whether multiple mechanisms exist which
relate to phylogenetic origin, the nature of the transported
substrate and the direction of transport. However, based
on the data mentioned above we may at least consider
the possibility that the transport process of ABC
transporters involves two general drug transport sites
which, in multidrug transporters, may be composed of
multiple drug interaction sites to account for the wide range
of compounds that are transported. Transport may
potentially occur through or along a proteinacious chamber
spanning the phospholipid bilayer in which aromatic
residues and the protein-lipid interface may provide a “slide
guide” system for hydrophobic drugs. Recent
pharmacological studies on P-glycoprotein suggest that,
in addition to drug transport sites, regulatory binding sites
exist which may reside outside of the drug transport sites,
to which allosteric modulators bind which are not
transported themselves (Martin et al., 1999, 2000). Detailed
knowledge about the molecular interactions that occur
between drugs, modulators and their binding sites, and
the localization of these sites in the transport proteins will
ultimately require the determination of the threedimensional protein structure. A recent report of Pglycoprotein at 2.5 nm resolution already showed a
monomeric molecule with discrete domains (Rosenberg
et al., 1997). For LmrA, milligram quantities of highly purified
protein can be obtained rather easily which facilitates
current protein crystallization trials.
Acknowledgements
This research was funded by the EU program on Structural Biology, the
Dutch Cancer Society, the Medical Research Council, and Cancer Research
Campaign.
References
Ambudkar, S.V., Cardarelli, C.O., Pashinsky, I., and Stein, W.D. 1997.
Relation between the turnover number for vinblastine transport and for
vinblastine-stimulated ATP hydrolysis by human P-glycoprotein. J. Biol.
Chem. 272: 21160-21166.
Ambudkar, S.V., Dey, S., Hrycyna, C.A., Ramachandra, M., Pastan, I., and
Gottesman, M.M. 1999. Biochemical, cellular, and pharmacological
aspects of the multidrug transporter. Annu. Rev. Pharmacol. Toxicol. 39:
361-398.
Ayesh, S., Shao, Y.-M., and Stein, W.D. 1996. Coopertive, competitive and
non-competitive interactions between modulators of P-glycoprotein.
Biochim. Biophys. Acta 1316: 8-18.
Bauer, B.E., Wolfger, H., and Kuchler, K. 2000. Inventory and function of
yeast ABC proteins: about sex, stress, pleiotropic drug and heavy
metal resistance. Biochim Biophys Acta. 1461: 217-236.
Bolhuis, H., van Veen, H.W., Molenaar, D., Poolman, B., Driessen, A.J.M.,
and Konings, W.N. 1996a. Multidrug resistance in Lactococcus lactis:
evidence for ATP-dependent drug extrusion from the inner leaflet of the
cytoplasmic membrane. EMBO J. 15: 4239-4245.
Bolhuis, H., van Veen, H.W., Brands, J.R., Putman, M., Poolman, B.,
Driessen, A.J.M., and Konings, W.N. 1996b. Energetics and mechanism
of drug transport by the lactococcal multidrug transporter LmrP. J. Biol.
Chem. 271: 24123-24128.
Bruggeman, E.P., Currier, S.J., Gottesman, M.M., and Pastan, I. 1992.
Characterization of the azidopine and vinblastine binding site of Pglycoprotein. J. Biol. Chem. 267: 21020-21026.
Busche, R., Tümmler, B., Cano-Gauchi, D.F., and Riordan, J.R. 1989.
Equilibrium, kinetic and photoaffinity labeling studies of daunomycin
binding to P-glycoprotein-containing membranes of multidrug-resistant
Chinese hamster ovary cells. Eur. J. Biochem. 183: 189-197.
Chiba, P., Ecker, G., Schmid, D., Drach, J., Tell, B., Goldenberg, S., and
Gekeler, V. 1996. Structural requirements for activity of propafenone-type
modulators in P-glycoprotein-mediated multidrug resistance. Mol.
Pharmacol. 49: 1122-1130.
Cole, S.P.C., and Deeley, R.G. 1998. Multidrug resistance mediated by the
ATP-binding cassette transporter protein MRP. BioEssays 20: 931-940.
Demmer, A., Thole, H., Kubesch, P., Brandt, T., Raida, M., Fislage, R., and
Tümmler, B. 1997. Localization of the iodomycin binding site in hamster
P-glycoprotein. J. Biol. Chem. 272: 20913-20919.
Döppenschmitt, S., Langguth, P., Regårdh, C.G., Andersson, T.B.,
Hilgendorf, C., and Spahn-langguth. 1999. Characterization of binding
properties to human P-glycoprotein: development of a [ 3H]verapamil
radioligand-binding assay. J. Pharmacol. Exp. Therap. 288: 348-356.
Dougherty, D.A. 1996. Cation-π interactions in chemistry and biology: a
new view of benzene, Phe, Tyr, and Trp. Science 271: 163-168.
Dey, S., Ramachandra, M., Pastan, I., Gottesman, M., and Ambudkar,
S.V. 1997. Evidence for two non-identical drug-interaction sites in the
human P-glycoprotein. Proc. Natl. Acad. Sci. USA 94: 10594-10599.
Ecker, G., Huber, M., Schmid, D., and Chiba, P. 1999. The importance of a
nitrogen atom in modulators of multidrug resistance. Mol. Pharmacol. 56:
791-796.
Edgar, R., and Bibi, E. 1999. A single membrane-embedded negative charge
is critical for recognizing positively charged drugs by the Escherichia coli
multidrug resistance protein MdfA. EMBO J. 1999 18: 822-832.
Ehrmann, M., Ehrle, R., Hofmann, E., Boos, W., and Schlösser, A. 1998.
The ABC maltose transporter. Mol. Microbiol. 29: 685-694.
Evers, R., de Haas, M., Sparidans, R., Beijnen, J., Wielinga, P.R., Lankelma,
J., and Borst, B. 2000. Vinblastine and sulfinpyrazone export by the
multidrug resistance protein MRP2 is associated with glutathione export.
Br. J. Canc. 83: 375-383.
Eytan, G.D., Regev, R., and Assaraf, Y.G. 1996. Functional reconstitution
of P-glycoprotein reveals an apparent near stoichiometric drug transport
to ATP hydrolysis. J. Biol. Chem. 271: 3172-3178.
Frezard, F., and Garnier-Suillerot, A. 1991. DNA-containing liposomes as a
model for the study of cell membrane permeation by antracycline
derivatives. Biochemistry 30: 5038-5043.
Gadsby, D.C., and Nairn, A.C. 1999. Control of CFTR channel gating by
phosphorylation and nucleotide hydrolysis. Physiol. Rev. 79: S77-S107.
Gottesman, M.M., Hrycyna, C.A., Schoenlein, P.V., Germann, U.A., and
Pastan, I. 1995. Genetic analysis of the multidrug transporter. Annu. Rev.
Genet. 29: 607-649.
Greenberger, L.M. 1993. Major photoaffinity drug labeling sites for
iodoarylazidoprazosin in P-glycoprotein are within, or immediately Cterminal to, transmembrane domains 6 and 12. J. Biol. Chem. 268: 1141711425.
van der Heide, T., and Poolman, B. 2000. Osmoregulated ABC-transport
system of Lactococcus lactis senses water stress via changes in the
physical state of the membrane. Proc. Natl. Acad. Sci. USA 20: 71027106.
Higgins, C.F. 1992. ABC transporters: from microorganisms to man. Annu.
Rev. Cell Biol. 8: 67-113.
Higgins, C.F., and Gottesman, M.M. 1992. Is the multidrug transporter a
flippase? TIBS 17: 18-21.
Higgins, C.F., Callaghan, R., Linton, K.J., Rosenberg, M.F., and Ford, R.C.
1997. Structure of the multidrug resistance P-glycoprotein. Sem. Canc.
Biol. 8: 135-142.
Hipfner, D.R., Deeley, R.G., and Cole, S.P.C. 1999. Structural, mechanistic
and clinical aspects of MRP1. Biochim. Biophys. Acta 1461: 359-376.
Holland, B., and Blight, M.A. 1999. ABC-ATPases, adaptable energy
generators fuelling transmembrane movement of a variety of molecules
in organisms from bacteria to humans. J. Mol. Biol. 293: 381-399.
Homolya, L., Holl, Z., Germannn, U.A., Pastan, I., Gottesman, M.M., and
Sarkadi, B. 1993. Fluorescent cellular indicators are extruded by the
multidrug resistance protein. J. Biol. Chem. 268: 21493-21496.
Kast, C., Canfield, V., Levenson, R., and Gros, P. 1996. Transmembrane
organisation of mouse P-glycoprotein determined by epitope insertion
and immunofluorescence. J. Biol. Chem. 271: 9240-9248.
Klopman, G., Shi, L.M., Ramu, 1997. Quantitative structure-activity
relationship of multidrug resistance reversal agents. Mol. Pharmacol. 52:
323-334.
Kolaczkowski, M., van der Rest, M., Cybularz-Kolaczkowski, A., Soumillion,
J.-P., Konings, W.N., and Goffeau, A. 1996. Anticancer drugs, ionophoric
peptides, and steroids as substrates of the yeast multidrug transporter
Pdr5p. J. Biol. Chem. 271: 31543-31548.
König, J., Nies, A.T., Cui, Y., Leier, I., and Keppler, D. 1999. Conjugate
export pumps of the multidrug resistance protein (MRP) family: localization,
substrate specificity, and MRP2-mediated drug resistance. Biochim.
Biophys. Acta 1461: 377-394.
192 van Veen et al.
Kwan, T., Loughrey, H., Brault, M., Gruenheid, S., Urbatsch, I.L., Senior,
A.E., and Gros, P. 2000. Functional analysis of a tryptophan-less Pglycoprotein: a tool for tryptophan insertion and fluorescence spectroscopy.
Mol. Pharmacol. 58: 37-47.
Liu, C.E., Liu, P.-Q., Wolf, A., Lin, E., and Ames, G.F.-L. 1998. Both lobes of
the soluble receptor of the periplasmic histidine permease, an ABC
transporter (Traffic ATPase), interact with the membrane-bound complex.
J. Biol. Chem. 274: 739-747.
Loe, D.W., Deeley, R.G., and Cole, P.C. 2000a. Verapamil stimulates
glutathione transport by the 190-kDa multidrug resistance protein 1
(MRP1). J. Pharmacol. Exp. Therapeut. 293: 530-538.
Loe, D.W., Deeley, R.G., and Cole, P.C. 2000b. Characterization of
vincristine transport by the Mr 190,000 multidrug resistance protein (MRP):
evidence for cotransport with reduced glutathione. Canc. Res. 58: 51305136.
Loo, T.W., and Clarke, D.M. 1999. Determining the structure and mechanism
of the human multidrug resistance P-glycoprotein using cysteine-scanning
mutagenesis and thiol-modification techniques. Biochim. Biophys. Acta
1461: 315-325.
Martin, C., Berridge G., Higgins, C.F., Mistry, P., Charlton, P., and Callaghan,
R., 2000. Communication between multiple drug binding sites on Pglycoprotein. Mol. Pharmacol. 58: 624-632.
Martin, C., Berridge G., Mistry, P., Higgins, C.F., Charlton, P., and Callaghan,
R., 1999. The molecular interaction of the high affinity reversal agent
XR9576 with P-glycoprotein. Br. J. Pharmacol. 128: 403-411.
Mechetner, E.B., Schott, B., Morse, B.S., Stein, W.D., Druley, T., Davis,
K.A., Tsuruo, T., and Roninson, I.B. 1997. P-glycoprotein function involves
conformational transitions detectable by differential immunoreactivity.
Proc. Natl. Acad. Sci. USA 94: 12908-12913.
Muth, T.R., and Schuldiner, S. 2000. A membrane-embedded glutamate is
required for ligand binding to the multidrug transporter EmrE. EMBO J.
19: 234-240.
Neyfakh, A.A., Bidnenko, V.E., and Chen, L.B. 1991. Efflux-mediated
multidrug resistance in Bacillus subtilis: similarities and dissimilarities with
the mammlian system. Proc. Natl. Acad. Sci. USA 88: 4781-4785.
Orlowski, S., and Garrigos, M. 1999. Multiple recognition of various
amphiphilic molecules by the multidrug resistance P-glycoprotein:
molecular mechanisms and pharmacological consequences coming from
functional interactions between various drugs. Anticanc. Res. 19: 31093124.
Pajeva, I.K., and Wiese, M. 1998. A comparative molecular field analysis of
propafenone-type modulators of cancer multidrug resistance. Quant.
Struct.-Act. Relat. 17: 301-312.
Park, Y., Cho, Y.-J., Ahn, T., and Park, C. 1999. Molecular interactions in
ribose transport: the binding protein module symmetrically associates
with the homodimeric membrane transporter. EMBO J. 18: 4149-4156.
Paulsen, I.T., Brown, M.H., and Skurray, R.A. 1996. Proton-dependent
multidrug efflux systems. Microbiol. Rev. 60: 575-608.
Prasad, R., de Wergifosse, P., Goffeau, A., and Balzi, E. 1995. Molecular
cloning and characterization of a novel gene of Candida albicans, CDR1,
conferring multiple resistance to drugs and antifungals. Curr. Genet. 27:
320-329.
Putman, M., van Veen, H.W., Degener, J.E., and Konings, W.N. 2000.
Antibiotic resistance: era of the multidrug pump. Mol. Microbiol. 36: 772774.
Ramachandra, M., Ambudkar, S.V., Chen, D., Hrycyna, C.A., Dey, S.,
Gottesman, M.M., and Pastan, I. 1998. Human P-glycoprotein exhibits
reduced affinity for substrates during a catalytic transition state.
Biochemistry 37: 5010-5019.
Raviv, Y., Pollard, H.B., Bruggeman, E.P., Pastan, I., and Gottesman, M.M.,
1990. Photosensitized labeling of a functional multidrug transporter in
living drug resistant tumor cells. J. Biol. Chem. 265: 3975-3980.
Rosenberg, M.F., Callaghan, R., Ford, R.C., and Higgins, C.F. 1997.
Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution
determined by electron microscopy and image analysis. J. Biol. Chem.
272: 10685-10694.
Seelig, A. 1998. A general pattern for substrate recognition by P-glycoprotein.
Eur. J. Biochem. 251: 252-261.
Seelig, A., Blatter, L., and Wohnsland. 2000. Substrate recognition by Pglycoprotein and the multidrug resistance-associated protein MRP1: a
comparison. Int. J. Clin. Pharmacol. Therap. 38: 111-121.
Senior, A.E., and Bhagat, S. 1998. P-glycoprotein shows strong catalytic
cooperativity between the two nucleotide sites. Biochemistry 37: 831836.
Senior, A.E., and Gadsby, D.C. 1997. ATP hydrolysis and mechanism in Pglycoprotein and CFTR. Sem. Canc. Biol. 8: 143-150.
Senior, A.E., Al-Shawi, M.K., and Urbatsch, I.L. 1995. The catalytic cycle of
P-glycoprotein. FEBS Lett. 377: 285-289.
Shapiro, A.B., and Ling, V. 1997a. Extraction of Hoechst 33342 from the
cytoplasmic leaflet of the plasma membrane by P-glycoprotein. Eur. J.
Biochem. 250: 122-129.
Shapiro, A.B., and Ling, V. 1997b. Positively cooperative sites for drug
transport by P-glycoprotein with distinct drug specificities. Eur. J. Biochem.
250: 130-137.
Shapiro, A.B., and Ling, V. 1998. Transport of LDS-751 from the cytoplasmic
leaflet of the plasma membrane by the rhodamine-123-selective site of
P-glycoprotein. Eur. J. Biochem. 254: 181-188.
Shapiro, A.B., Corder, A.B., and Ling, V. 1997. P-glycoprotein-mediated
Hoechst 33342 transport out of the lipid bilayer. Eur. J. Biochem. 250:
115-121.
Sharom, F.J., Liu, R., Romsicki, Y., and Lu, P. 1999. Insights into the structure
and substrate interactions of the P-glycoprotein multidrug transporter from
spectroscopic studies. Biochim. Biophys. Acta 1461: 327-344.
Sharom, F.J., Yu, X., Didiodato, G., and Chu, J.W.K. 1996. Synthetic
hydrophobic peptides are substrates for P-glycoprotein and stimulate drug
transport. Biochem. J. 320: 421-428.
Sonveaux, N., Shapiro, A.B., Goormaghtigh, E., Ling, V., and Ruysschaert,
J.M. 1996. Secondary and tertiary structure changes of reconstituted Pglycoprotein. J. Biol. Chem. 271: 24617-24624.
Sonveaux, N., Vigano, C., Shapiro, A.B., Ling, V., and Ruysschaert, J.M.
1999. Ligand-mediated tertiary structure changes of reconstituted Pglycoprotein. J. Biol. Chem. 274: 17649-17654.
Speelmans, G., Staffhorst R.W.H.M., Steenbergen, H.G., and de Kruijff, B.
1996. Transport of the anti-cancer drug doxorubicin across cytoplasmic
membrane and membranes composed of phospholipids derived from
Escherichia coli occurs via a similar mechanism. Biochim. Biophys. Acta
1284: 240-246.
Stride, B.D., Cole, S.P.C., and Deeley, R.G. 1999. Localization of a substrate
specificity domain in the multidrug resistance protein. J. Biol. Chem. 274:
22877-22883.
Taglicht, D., and Michaelis, S. 1998. Saccharomyces cerevisiae ABC
proteins and their relevance to human health and disease. Methods
Enzymol. 292: 101-116.
Tanford. C. 1983. Translocation pathway in the catalysis of active transport.
Proc. Natl. Acad. Sci. USA 80: 3701-3705.
Tusnády, G.E., Bakos, E., Varadi, A., and Sarkadi, B. 1997. Membrane
topology distinguishes a subfamily of the ATP-binding cassette (ABC)
protein superfamily. EMBO J. 10: 3777-3785.
Ueda, K., Matsuo, M., Tanabe, K., Morita, K., Kioka, N., and Amachi, T.
1999. Comparative aspects of the function and mechanism of SUR1 and
MDR1 proteins. Biochim. Biophys. Acta 1461: 305-313.
Ueda, K., Taguchi, Y., and Morishima, M. 1997. How does P-glycoprotein
recognize its substrate? Sem. Canc. Biol. 8: 151-159.
Urbatsch, I.L., Sankaran, B., Weber, J., and Senior, A.E. 1995. Pglycoprotein is stably inhibited by vanadate-induced trapping of nucleotide
at a single catalytic site. J. Biol. Chem. 270: 19383-19390.
Urbatsch, I.L., Beaudet, L., Carrier, I., and Gros, P. 1998. Mutations in either
nucleotide-binding site of P-glycoprotein (Mdr3) prevent vanadate trapping
of nucleotide at both sites. Biochemistry 37: 4592-4602.
van Veen, H.W., Callaghan, R., Soceneantu, L., Sardini, A., Konings, W.N.,
and Higgins, C.F. 1998. A bacterial antibiotic-resistance gene that
complements the human multidrug-resistance P-glycoprotein gene. Nature
391: 291-295.
van Veen, H.W., Margolles, A., Müller, M., Higgins, C.F., and Konings, W.N.
2000. The homodimeric ATP-binding transporter LmrA mediates multidrug
transport by an alternating two-site (two-cylinder engine) mechanism.
EMBO J. 19: 2503-2514.
van Veen, H.W., Venema, K., Bolhuis, B., Oussenko, I., Kok, J., Poolman,
B., Driessen, A.J.M., and Konings, W.N. 1996. Multidrug resistance
mediated by a bacterial homolog of the human drug transporter MDR1.
Proc. Natl. Acad. Sci. USA 93: 10668-10672.
Vázquez-Laslop, N., Markham, M., Neyfakh, A.A. 1999. Mechanism of ligand
recognition by BmrR, the multidrug-responding transcriptional regulator:
mutational analysis of the ligand-binding site. Biochemistry 38: 1692516931.
Vigano, C., Margolles, A., van Veen, H.W., Konings, W.N., and Ruysschaert,
J.-M. 2000. Secondary and tertiary structure changes of reconstituted
LmrA induced by nucleotide binding or hydrolysis. J. Biol. Chem. 275:
10962-10967.
Wang, G., Pincheira, R., and Zhang, J-.T. 1998. Dissection of drug-bindinginduced conformational changes in P-glycoprotein. Eur. J. Biochem. 255:
383-390.
Yerushalmi, H., and Schuldiner, S. 2000. A common site for substrates and
protons in EmrE, an ion-coupled multidrug transporter. J. Biol. Chem.
275: 5264-5269.
Zheleznova, E.E., Markham, P.N., Neyfakh, A.A., and Brennan, R.G. 1999.
Structural basis of multidrug recognition by BmrR, a transcription activator
of a multidrug transporter. Cell 96: 353-362.
Zheleznova, E.E., Markham, P., Edgar, R., Bibi, E., Neyfakh, A.A., and
Brennan, R.G. 2000. A structure-based mechanism for drug binding by
multidrug transporters. TIBS 25: 39-43.