From the Canadian Institutes for Health Research Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada
Received for publication, November 5, 2002
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ABSTRACT |
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The human multidrug resistance
P-glycoprotein (P-gp, ABCB1) uses ATP to transport many structurally
diverse compounds out of the cell. It is an ABC transporter with two
nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs).
Recently, we showed that the "LSGGQ" motif in one NBD
(531LSGGQ535 in NBD1;
1176LSGGQ1180 in NBD2) is adjacent to the
"Walker A" sequence
(1070GSSGCGKS1077 in NBD2;
427GNSGCGKS434 in NBD1) in the other NBD (Loo,
T. W., Bartlett, M. C., and Clarke, D. M. (2002)
J. Biol. Chem. 277, 41303-41306). Drug substrates can
stimulate or inhibit the ATPase activity of P-gp. Here, we report the
effect of drug binding on cross-linking between the LSGGQ signature
and Walker A sites (Cys431(NBD1)/C1176C(NBD2) and
Cys1074(NBD2)/L531C(NBD1), respectively). Seven drug
substrates (calcein-AM, demecolcine, cis(Z)-flupentixol,
verapamil, cyclosporin A, Hoechst 33342, and
trans(E)-flupentixol) were tested for their effect on
oxidative cross-linking. Substrates that stimulated the ATPase activity
of P-gp (calcein-AM, demecolcine, cis(Z)-flupentixol, and
verapamil) increased the rate of cross-linking between
Cys431(NBD1-Walker A)/C1176C(NBD2-LSGGQ) and between
Cys1074(NBD2-Walker A)/L531C(NBD1-LSGGQ) when compared with
cross-linking in the absence of drug substrate. By contrast, substrates
that inhibited ATPase activity (cyclosporin A, Hoechst 33342, and
trans(E)-flupentixol) decreased the rate of cross-linking.
These results indicate that interaction between the LSGGQ motifs and
Walker A sites must be essential for coupling drug binding to ATP
hydrolysis. Drug binding in the transmembrane domains can induce long
range conformational changes in the NBDs, such that compounds that
stimulate or inhibit ATPase activity must decrease and increase,
respectively, the distance between the Walker A and LSGGQ sequences.
P-glycoprotein
(P-gp)1 is an
ATP-dependent drug pump that transports numerous
structurally diverse compounds of different sizes out of the cell
(recently reviewed in Refs. 1 and 2). Therefore, P-gp can complicate
cancer and AIDS chemotherapy because many therapeutic compounds are
substrates of P-gp (3, 4).
P-gp is a single polypeptide of 1280 amino acids. It is organized as
two repeating units of 610 amino acids that are joined by a linker
region of about 60 amino acids (5). Each repeat has six transmembrane
(TM) segments and a hydrophilic domain containing an ATP-binding site
(6, 7). P-gp functions as a monomer (8), but the two halves of the
molecule do not have to be covalently linked to function (9,
10). The transmembrane domains alone are sufficient to mediate
drug binding (10), but both ATP-binding sites must be functional for
drug efflux activity (11-14).
An important aspect in understanding the mechanism of P-gp is how drug
transport is coupled to ATP hydrolysis. The observation that drug
binding to P-gp can either stimulate or inhibit ATP hydrolysis suggests
that drug binding and ATP hydrolysis must be tightly regulated (12, 15,
16).
The "signature" sequence (LSGGQ) in each NBD appears to be an
important region in P-gp. Although the signature sequences are present
in all ABC transporters (17), their function is unknown. We recently
showed that the LSGGQ in one NBD was close to the Walker A sequence in
the other NBD (18). We postulated that the LSGGQ sequence might play a
role in conveying conformational changes from the drug-binding site to
the ATP-binding sites. In this study, we examined the effect of drug
substrates on cross-linking between the LSGGQ motifs and the Walker A sites.
Construction of Mutants--
A histidine-tagged Cys-less P-gp
was constructed and then used for making mutants containing pairs of
cysteines (6, 19, 20). Two mutants that contained a cysteine in the
LSGGQ site and another in the Walker A site were constructed (18). One mutant (L531C/Cys1074) contained a cysteine in the
NH2-terminal 531LSGGQ535 site and
an endogenous cysteine (Cys1074) in the COOH-terminal
Walker A site (1070GSSGCGKC1077). The other
mutant (Cys431/L1176C) contained the endogenous
Cys431 in the NH2-terminal Walker A site
(427GNSGCGKS434) and another cysteine in the
COOH-terminal 1176LSGGQ1180 site.
Expression, Disulfide Cross-linking Analysis, and
Purification--
The mutant cDNAs were expressed in HEK 293 cells
in the presence of cyclosporin A to promote maturation of P-gp (21,
22). Membranes were prepared as described previously (19, 23). For
disulfide cross-linking analysis, aliquots of membranes were added to
equal volumes of Tris-buffered saline (10 mM Tris-HCl, pH
7.4, 150 mM NaCl) containing 1 mM
Cu2+-(phenanthroline)3. The samples were
incubated at 21 or 4 °C for various intervals, and the reactions
were stopped by the addition of SDS sample buffer (125 mM
Tris-HCl, pH 6.8, 20% (v/v) glycerol, and 4% (w/v) SDS) containing 50 mM EDTA and no reducing agent. The reaction mixtures were
subjected to SDS-PAGE (7.5% polyacrylamide gels) and immunoblot
analysis with a rabbit polyclonal antibody against P-gp (8).
To test the effect of nucleotide or vanadate on cross-linking, the
membranes were incubated with an equal volume of Tris-buffered saline
containing the following: 1) 12 mM ATP, 24 mM
MgCl2 and 0.6 mM sodium orthovanadate; 2) 12 mM ATP and 24 mM MgCl2; 3) 12 mM ATP; 4) 24 mM MgCl2; 5) 0.6 mM sodium orthovanadate; 6) 12 mM ADP; or 7) 12 mM AMP-PNP. Sodium orthovanadate was prepared from
Na3VO4, pH 10 (24), and boiled for 2 min to
break down polymeric species (25). The samples were incubated for 10 min at 37 °C and then cooled in an ice bath before treatment with
oxidant at 21 °C. At this temperature there is almost complete
cross-linking in both mutants (18).
To test the effect of drug substrates on cross-linking, the mutant
P-gps were preincubated with drug substrate for 10 min at 21 °C,
then chilled at 4 °C for 10 min, and then treated with oxidant. At
4 °C, the rate of cross-linking is also slowed, and this allowed us
to detect changes in cross-linking.
The purification of histidine-tagged P-gp mutants and an assay of
drug-stimulated ATPase activities were done as described previously
(23, 26) except that the isolated samples were mixed with
Escherichia coli lipid rather than sheep brain
phosphatidylethanolamine. E. coli lipids were used because
basal P-gp ATPase activity is higher in these lipids than those with
sheep brain phosphatidylethanolamine. This made measuring the
inhibition of P-gp ATPase activity much easier. Also, the
drug-stimulated ATPase activity of P-gp reconstituted with E. coli lipids is similar to that measured in isolated mammalian plasma membranes that are enriched in P-gp (15).
We showed previously by disulfide cross-linking analysis that the
contact between the NBDs of P-gp could occur between the LSGGQ
signature sequence in one NBD and the Walker A site in the other NBD
(18). Mutants in which the leucine residue in the LSGGQ site is
replaced with cysteine can be oxidatively cross-linked with the
endogenous cysteine in the opposing Walker A sequence ((L531C(NBD1-LSGGQ)/Cys1074(NBD2-Walker A) or
Cys431(NBD1-Walker A)/L1176C(NBD2-LSGGQ)). Fig.
1 shows that that cross-linking was
almost complete in mutants L531C/Cys1074 and
Cys431/L1176C when treated with 0.5 mM copper
phenanthroline for 15 min at 21 °C. We previously showed that
cross-linking occurred in the active molecule, because cross-linking
resulted in an inactive molecule. Activity was restored, however, after
the addition of dithiothreitol (18).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cross-linking of P-gp mutants. Membranes
were prepared from HEK 293 cells expressing mutants
L531C/Cys1074 (L531C/C1074) or
Cys431/L1176C (C431/L1176C). The
membranes were preincubated with no additions (None) or with
Mg-ATP plus vanadate (Mg.ATP+Vi), ATP, MgCl2
(Mg), vanadate (Vi), MgCl2 plus ATP
(Mg.ATP), ADP, or AMP-PNP (AMP.PNP) for 10 min at
37 °C. The membranes were then treated with oxidant for 15 min at
21 °C. The reactions were stopped by the addition of SDS sample
buffer containing 50 mM EDTA and no reducing agent. The
mixtures were subjected to immunoblot analysis. The positions of the
cross-linked (X-link) product and mature (170 kDa) P-gp are indicated. CuP, copper
phenanthroline.
To determine whether cross-linking between the NBDs could be disrupted,
the mutants were pre-treated with nucleotide or subjected to vanadate
trapping. P-gp traps nucleotides in the presence of vanadate plus
Mg-ATP and results in a transition state (27, 28). Vanadate traps ADP
at either NBD by occupying the position of the -phosphate adjacent
to ADP. Vanadate trapping at one site then inhibits ATP hydrolysis at
the second ATP-binding site (27). Fig. 1 shows that inhibition of
cross-linking in mutants L531C/Cys1074 and
Cys431/L1176C was observed only after treatment with
vanadate plus Mg-ATP. Inhibition of cross-linking was not observed when
the mutants were pre-treated with ATP, MgCl2, vanadate,
Mg-ATP, ADP, or with the non-hydrolyzable ATP analog AMP-PNP. It is
unlikely that vanadate trapping of nucleotide denatures the protein,
because trapping of nucleotide is reversible (27).
We have proposed that the LSGGQ motifs might participate in transmitting conformational changes from the transmembrane domains to the NBDs (18). Mutations in the LSGGQ motifs do not prevent ATP binding or vanadate trapping of nucleotides (29, 30). One way of inducing different conformational changes in the TMDs is to use drug substrates with different structures. P-gp is interesting in that some drug substrates stimulate whereas others inhibit the ATPase activity of P-gp. Therefore, it is possible that the conformational changes in the TMDs may be monitored by changes in the cross-linking patterns in mutants L531C/Cys1074 and Cys431/L1176C.
Drug substrates (1) that either stimulated (calcein-AM, demecolcine,
cis(Z)-flupentixol, and verapamil) or inhibited (cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) the ATPase
activity of Cys-less P-gp (data not shown) were identified. The
flupentixol isomers are interesting in that the
cis(Z)-isomer stimulates, whereas the
trans(E)-isomer inhibits the ATPase activity of wild-type P-gp (31). These drug substrates were then tested on mutants L531C/Cys1074 and Cys431/L1176C. Fig.
2 shows that the ATPase activity of
mutant L531C/Cys1074 was stimulated by calcein-AM,
demecolcine, cis(Z)-flupentixol, and verapamil (6.4-, 6.8-, 3.5-, and 4-fold, respectively). Half-maximal stimulation of ATPase
activity with calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil occurred at 30, 163, 29, and 9 µM,
respectively. Cyclosporin A, Hoechst 33342, and
trans(E)-flupentixol inhibited the activity of mutant
L531C/Cys1074 with 50% inhibition occurring at
concentrations of about 0.12, 0.67, and 1.1 mM,
respectively. Similar results were obtained with mutant
Cys431/L1176C (data not shown).
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We then tested whether the structurally different stimulatory and
inhibitory drug substrates affected cross-linking in the NBDs.
Accordingly, membranes from mutant L531C/Cys1074 were
preincubated for 10 min at 21 °C with 1 mM calcein-AM, 2 mM demecolcine, 1 mM
cis(Z)-flupentixol, 0.2 mM verapamil, 0.5 mM cyclosporin A, 0.5 mM Hoechst 33342, or 1 mM trans(E)-flupentixol. These concentrations
were required for maximal stimulation or inhibition of ATPase activity
(Fig. 2). The membranes were then treated with oxidant at 4 °C for
various intervals. The rationale for doing the cross-linking at 4 °C
was that thermal motion in the protein would be reduced and that subtle
changes caused by drug substrate binding may be detected. Fig.
3 shows the effects of substrates on
cross-linking of mutant L531C/Cys1074. In the absence of
drug substrates, ~50% of the mutant protein was cross-linked by 16 min. In the presence of compounds (calcein-AM, demecolcine,
cis(Z)-flupentixol, and verapamil) that stimulate the ATPase
activity of P-gp, however, the rate of cross-linking was significantly
increased so that 50% cross-linking occurred by 2 min. The presence of
the inhibitory compounds (cyclosporin A, Hoechst 33342, and
trans(E)-flupentixol) had the opposite effect. In the
presence of these inhibitors, <50% cross-linking was observed at 32 min (Fig. 3).
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We then tested the effect of drug substrates on cross-linking of mutant
Cys431/L1176C. In the absence of drug substrates, 50% of
the mutant protein was cross-linked with an oxidant after 16 min (Fig.
4). In the presence of the stimulatory
drug substrates (calcein-AM, demecolcine,
cis(Z)-flupentixol, and verapamil), the rate of
cross-linking was increased, because 50% of the mutant protein was
cross-linked by 2-4 min. Drug substrates that inhibited ATPase
activity (cyclosporin A, Hoechst 33342, and
trans(E)-flupentixol) of the mutant P-gp also inhibited the
cross-linking of the mutant protein. Fig. 4 shows that in the presence
of these compounds <50% cross-linking occurred at 32 min.
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The effect of substrates on mutants L531C/Cys1074 and
Cys431/L1176C were very similar. Compounds that stimulated
the ATPase activity also stimulated the rate of cross-linking of the
mutants, whereas those that inhibited ATPase activity also inhibited
the rate of cross-linking. It is unlikely that drug substrates are
binding directly to the ATP-binding sites, because it has been shown
that drug binding does not alter the affinity for ATP (15, 16, 32).
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DISCUSSION |
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Disulfide cross-linking between adjacent cysteines in the Walker A sequence of one NBD and the LSGGQ site in the other NBD is a useful approach for monitoring changes in the NBDs. A condition that dramatically affects cross-linking between these two sites occurs after vanadate trapping of a nucleotide (Fig. 1). Hydrolysis of ATP in the presence of vanadate was essential for the inhibition of cross-linking because cross-linking was not inhibited in the presence of the non-hydrolyzable ATP analog AMP-PNP. Similarly, cross-linking was not inhibited when the mutants were pre-incubated with only ATP, MgCl2, ADP, vanadate, or Mg-ATP. These results indicate that the trapped vanadate either occupies the space between the cross-linkable cysteines in the LSGGQ and Walker A sites in the two NBDs or that its presence causes the two NBDs to move apart.
The drug-binding site in the TMDs (33, 34) and the ATP binding sites in P-gp must be quite far apart. Fluorescence resonance energy transfer studies indicate that the ATP-binding sites are about 40 Å from the drug-binding site (35). Our results show that binding of drug substrates must induce conformational changes in the drug-binding site that are transmitted distally to the NBDs. Similarly, conformational changes in the NBDs are also transmitted to the drug-binding site in the TMDs (25, 28). Therefore, there must be continuous "cross-talk" among the domains of P-gp.
The LSGGQ sequence can increase or decrease the rate of ATP hydrolysis
depending on its distance from the Walker A site as shown in Fig.
5. Inhibitory substrates may cause both
of the Walker A and LSGGQ sites to move farther apart and/or reduce the
rate of ATP hydrolysis. In this study, the inhibitory substrates
reduced the rate of cross-linking, and this may occur by moving the two NBDs apart (Figs. 3 and 4). Drug substrates that stimulate ATPase activity of P-gp must bring the Walker A and LSGGQ sites closer together so that hydrolysis of ATP occurs at a faster rate.
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There is no detailed crystal structure information about eukaryotic ABC
transporters. Recent crystal structure studies on other ABC
transporters such as Rad50cd (36), BtuCD (37), and MJ0796 (38),
however, show that the LSGGQ sequences in these proteins are located
adjacent to the -phosphate of ATP. It is likely that as the LSGGQ
motif moves closer to the Walker A site, the rate of ATP hydrolysis
increases. Our results support this idea and may explain why a
substrate is a stimulator or inhibitor of P-gp. These results could
form the basis for the development of better inhibitors of
P-gp.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA80900 and by grants from the Canadian Institutes for Health Research (CIHR) and the Canadian Cystic Fibrosis Foundation.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.
Canada Research Chair in Membrane Biology and to whom
correspondence should be addressed: Dept. of Medicine, University of Toronto, Rm. 7342, Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel. or Fax: 416-978-1105; E-mail: david.clarke@utoronto.ca.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M211307200
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ABBREVIATIONS |
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The abbreviations used are:
P-gp, P-glycoprotein;
ABC, ATP-binding cassette;
AMP-PNP, adenosine
5'-(,
-imino)triphosphate;
NBD, nucleotide-binding domain;
NBD1, NH2-terminal NBD;
NBD2, COOH-terminal NBD;
TM, transmembrane;
TMD, TM domain.
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