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J. Biol. Chem., Vol. 275, Issue 26, 19435-19438, June 30, 2000

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ACCELERATED PUBLICATION
Drug-stimulated ATPase Activity of Human P-glycoprotein Is Blocked by Disulfide Cross-linking between the Nucleotide-binding Sites^*

Tip W. Loo and David M. Clarke

From the Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Received for publication, April 3, 2000, and in revised form, May 8, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

P-glycoprotein (P-gp) is an ATP-dependent drug pump that contains two nucleotide-binding domains (NBDs). Disulfide cross-linking analysis was done to determine if the two NBDs are close to each other. Residues within or close to the Walker A (GNSGCGKS in NDB1 and GSSGCGKS in NBD2) sequences for nucleotide binding were replaced with cysteine, and the mutant P-gps were subjected to oxidative cross-linking. Cross-linking was detected in two mutants, G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2), because the cross-linked proteins migrated slower in SDS gels. Mutants G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2) retained 10% and 82%, respectively, of the drug-stimulated ATPase activity relative to that of Cys-less P-gp. The cross-linking properties of the more active mutant L439C(NBD1)/Cys-1074(NBD2) were then studied. Cross-linking was reversed by addition of dithiothreitol and could be prevented by pretreatment of the mutant with N-ethylmaleimide. Cross-linking was also inhibited by MgATP, but not by the verapamil. Oxidative cross-linking of mutant L439C(NBD1)/Cys-1074(NBD2) resulted in almost complete inhibition of drug-stimulated ATPase activity. More than 60% of the drug-stimulated ATPase activity, however, was recovered after treatment with dithiothreitol. The results indicate that the two predicted nucleotide-binding sites are close to each other and that cross-linking inhibits ATP hydrolysis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The human multidrug resistance P-glycoprotein (P-gp)¹ is a plasma membrane protein that uses ATP to pump out of the cell a broad range of cytotoxic compounds that have diverse structures (1). Expression of P-gp is highest in the gastrointestinal tract, kidney, and liver and in the capillaries of the brain and testes where it may function to extrude endogenous and exogenous xenobiotics. Studies on "knock-out" mice suggest that this may be the function of P-gp (2).

P-gp consists of 1280 amino acids that are organized in two tandem repeats of 610 amino acids that are joined by a linker region of about 60 amino acids. Each repeat consists of an N-terminal hydrophobic domain containing six predicted transmembrane segments followed by a hydrophilic domain containing an ATP-binding site (3-5). The protein is a member of the ABC (ATP-binding cassette) family of transporters (6).

There has been considerable interest in determining the mechanism of transport by P-gp. Both halves of P-gp can hydrolyze ATP, but substrate-stimulated ATPase activity requires interaction between the two halves of the molecule (7). Similarly, both ATP-binding sites are essential because inactivation of either site by chemical modification (8-11) or mutagenesis (12, 13) results in loss of activity. It has been suggested that the nucleotide-binding sites function by an alternate site mechanism and show complete cooperativity (14).

An important aspect in understanding the mechanism of P-gp is the arrangement of the two NBDs. Recently, the crystal structure of the ATP-binding subunit (HisP) of the bacterial histidine permease complex was obtained and showed that the ATP-binding sites are oriented away from each other (15). There is evidence, however, that the two predicted ATP-binding sites of P-gp may be close to each other. It has been observed that dithiothreitol (DTT) causes activation of purified mouse mdr3 P-gp (16). Therefore, it was proposed that the endogenous cysteine residues in the Walker A homology sequences may form disulfide bonds. In this study, we tested whether cysteine residues introduced within or close to the Walker A homology sequences could be cross-linked.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of Mutants-- The construction of the cDNA for Cys-less P-gp (all seven endogenous cysteine residues change to alanine) was described previously (4). The cDNA was further modified to encode the epitope for monoclonal antibody A52 (4) or to encode a (His)₁₀ tag (13) at the COOH terminus of the molecule. The presence of an A52 tag facilitated detection of the mutant protein, whereas the histidine tag facilitated purification of the mutant P-gp by nickel-chelate chromatography. Cysteine residues were reintroduced into Cys-less P-gp cDNA by site-directed mutagenesis using synthetic oligonucleotides as described previously (17).

Disulfide Cross-linking, Purification of P-gp Mutants, and Measurement of Drug-stimulated ATPase Activity-- The cDNAs coding for the mutant P-gps were expressed in HEK 293 cells in the presence of 10 µM cyclosporin A as described previously (18). Expression of P-gp mutants in the presence of cyclosporin A promoted maturation of the protein (19). Cyclosporin A is a substrate of P-gp and acts as a chemical chaperone to enhance folding of P-gp. Membranes were prepared from HEK 293 cells expressing the mutant protein and subjected to oxidative cross-linking with 0.1 mM, 0.2 mM, or 1 mM Cu²⁺(phenanthroline)₃. The reactions were performed at 4, 22, or 37 °C for 10 min and then stopped by addition of EDTA to a final concentration of 25 mM. The samples were then treated with 5 mM N-ethylmaleimide to block unreacted thiol groups. The samples were then subjected to (5.5 or 6.5%) SDS-polyacrylamide gel electrophoresis, transferred onto a sheet of nitrocellulose, and probed with monoclonal antibody A52 or with rabbit polyclonal antibody against residues 439-640 of NBD1 (20), and the bands were visualized by enhanced chemiluminescence (Pierce).

The methods to test for the effects of cross-linking on ATPase activity were described previously (21). Briefly, membranes were prepared from 100 (10-cm diameter) culture plates of HEK 293 cells transfected with the mutant P-gp cDNA and grown with 10 µM cyclosporin A. Half of the membranes in Tris-buffered saline (TBS) buffer was incubated for 10 min at 37 °C in the presence of 0.1 mM Cu²⁺(phenanthroline)₃, and the other half was incubated for 10 min at 37 °C without oxidant. EDTA was then added to a final concentration of 25 mM. The samples were diluted 100-fold with TBS, and the membranes were collected by centrifugation at 45,000 × g for 45 min at 4 °C. The membranes were then solubilized with 1% (w/v) n-dodecyl--D-maltoside (Calbiochem), and histidine-tagged P-gp was recovered by nickel-chelate chromatography (Ni-NTA, Qiagen, Inc.) and assayed for verapamil-stimulated ATPase activity as described previously (13).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cross-linking of P-gp Mutants-- Wild-type P-gp has two cysteine residues within the predicted ATP-binding sites. Residue Cys-431 is in the Walker A consensus sequence in NBD1 (GNSGCGKS; also called the phosphate-binding loop or P-loop), whereas residue Cys-1074 is in the Walker A consensus sequence in NBD2 (GSSGGCGKS) (22). The P-loops are predicted to interact with the phosphate group of ATP (23). Following the P-loop is an -helix that is predicted to line the ATP-binding pocket.

To test if residues that are within or close to the P-loop of NBD1 are close to the P-loop of NBD2, we constructed a series of mutants containing a cysteine in NBD1 and a cysteine residue in NBD2 (Table I). One set of P-gp mutants contained Cys-431(NBD1) and a cysteine at positions 1069 to 1082 (NBD2), whereas another set of mutants had Cys-1074(NBD2) and a cysteine at positions 425 to 439 (NBD1). These mutants were expressed in HEK 293 cells, and membranes were prepared for oxidative cross-linking. We have shown that P-gp mutants containing disulfide cross-links between transmembrane segments in the two halves of the molecule migrate with slower mobility in SDS gels (18, 21, 24). It was therefore reasonable to assume that P-gp mutants with cross-links between the NBDs would also show altered mobility in SDS gels. Such an effect was also observed with the SERCA1 Ca²⁺-ATPase that has been cross-linked between the transmembrane segments (25) or within the NBD (26). In the absence of oxidant, mature P-gp migrates with an apparent mass of 170 kDa. Immature P-gp is also present in transfected HEK 293 cells and migrates with an apparent mass of 140 kDa. Cross-linking was detected in two mutants (Table I), G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2). The cross-linked product for both mutants migrated with slower mobilities in SDS gels after treatment with 0.2 mM Cu²⁺(phenanthroline)₃ for 10 min at 37 °C (Fig. 1). Cross-linked product was not detected for P-gp mutants containing one cysteine residue at positions 427, 439, or 1074 or in the Cys-less P-gp.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Oxidative cross-linking of P-gp. Membranes prepared from HEK 293 cells expressing A52-tagged Cys-less P-gp (C-less), mutant G427C(NBD1)/Cys-1074(NBD2) (G427C/C1074), mutant L439C(NBD1)/Cys-1074(NBD2) (L439C/C1074), or P-gp mutants containing one cysteine residue (G427C, L439C, or C1074) were treated with (+) or without () 0.2 mM Cu2+(phenanthroline)3 (oxidant) for 10 min at 37 °C. The reaction was stopped by addition of EDTA, and the samples were subjected to immunoblot analysis with monoclonal antibody A52, followed by enhanced chemiluminescence as described under "Experimental Procedures." The positions of the cross-linked (X-link) and mature (170-kDa) P-gp are indicated.

The results in Fig. 1 indicated that the two predicted ATP-binding sites were close to each other. It was important, however, to confirm that cross-linking was occurring in active mutants. Therefore, histidine-tagged P-gp mutants (G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2)) expressed in HEK 293 cells were purified by nickel-chelate chromatography. The purified proteins were mixed with lipid and sonicated, and the verapamil-stimulated ATPase activities were determined and compared with that of the Cys-less parent. Mutants (G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2)) had 10 and 82%, respectively, of the ATPase activity of Cys-less P-gp. Because mutant L439C(NBD1)/Cys-1074(NBD2) was more active, it was used for further analysis.

Effect of Temperature, Verapamil, DTT, and MalNEt on Cross-linking-- Mutant L439C(NBD1)/Cys-1074(NBD2) was then analyzed to see if cross-linking could be detected at lower temperatures or was affected by the presence of ATP, verapamil, DTT, or MalNEt. Fig. 2A shows that the cross-linked product was detected only at 37 °C.

View larger version (50K): [in this window] [in a new window]   Fig. 2.   Characterization of the cross-link in mutant L439C(NBD1)/Cys-1074(NBD2). Membranes were prepared from HEK 293 cells expressing A52-tagged mutant L439C(NBD1)/Cys-1074(NBD2) and treated as follows. A, the membranes were treated with (+) or without () 0.1 mM Cu2+(phenanthroline)3 (oxidant) for 10 min at 4, 22, or 37 °C. The reaction was stopped by the addition of EDTA, and the samples were subjected to immunoblot analysis with monoclonal A52. B, the membranes were preincubated with 10 mM MgATP (ATP) or 0.5 mM verapamil (Ver) for 5 min at 22 °C and then treated with (+) or without () oxidant for 10 min at 37 °C. The reaction was stopped by the addition of EDTA, and the samples were subjected to immunoblot analysis with monoclonal antibody A52. C, the membranes were treated with (+) or without () 0.1 mM oxidant for 10 min at 37 °C. The reaction was stopped by the addition of EDTA. The membranes were then treated with 10 mM DTT (+DTT). A sample of membranes was also treated with 5 mM N-ethylmaleimide (+NEM) before addition of oxidant. The reaction was stopped by the addition of EDTA, and the samples were subjected to immunoblot analysis with monoclonal antibody A52. D, the membranes were treated with 0.1 mM oxidant for 10 min at 37 °C, and the reaction was stopped by the addition of EDTA. The membranes were then incubated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin (final concentration, 10 µg/ml) for 5 min at 22 °C. The reaction was stopped by the addition of soybean trypsin inhibitor. Equivalent amounts were then incubated with (+) or without () 10 mM DTT. The samples were then subjected to immunoblot analysis with a mixture of monoclonal antibody A52 and rabbit polyclonal antibody against NBD1. The positions of the cross-linked (X-link) P-gp, mature (170-kDa) Pgp and proteolytic fragments (N-half) and (C-half) P-gp are indicated.

The membranes of mutant L439C(NBD1)/Cys-1074(NBD2) were then cross-linked in the presence of 10 mM MgATP or 1 mM verapamil. Verapamil was chosen as the drug substrate because it is the most potent stimulator of the ATPase activity of P-gp (27). Fig. 2B shows that the amount of cross-linked product was significantly reduced by the presence of ATP but not by the presence of verapamil. These results indicate that occupation of one or both nucleotide-binding sites blocks cross-linking.

In previous cross-linking studies, we confirmed that the change in mobility of P-gp after addition of oxidant was indeed due to cross-linking by repeating the experiments with the half-molecules (18). This was not possible with mutant L439C(NBD1)/Cys-1074(NBD2) because mutation L439C in the N-half molecule resulted in very low expression. We then used different approaches to show that the mobility shift after oxidative cross-linking of mutant L439C(NBD1)/Cys-1074(NBD2) was indeed due to disulfide bond formation.

Fig. 2C shows that the cross-linked product in mutant L439C(NBD1)/Cys-1074(NBD2) disappeared on addition of 10 mM DTT. This is consistent with the reduction of the disulfide bond. When membranes from mutant L439C(NBD1)/Cys-1074(NBD2) were pretreated with 5 mM N-ethylmaleimide before addition of oxidant, no cross-linked product was detected (Fig. 2C). It was shown that modification of either Cys-431(NBD1) or Cys-1074(NBD2) by N-ethylmaleimide inhibited the ATPase activity of P-gp (10, 11, 28). This result indicates that modification of Cys-1074 prevents disulfide bond formation.

We then examined the effect of trypsin on the cross-linked product. Trypsin cleaves P-gp in half because of one or more trypsin-sensitive sites within or close to the linker region (29, 30). If the two halves of P-gp are joined by a disulfide bond after oxidative cross-linking, then the release of the two halves of P-gp after trypsin treatment will require reduction of the disulfide bond. Fig. 2D shows that this was indeed the case. After trypsin treatment of the cross-linked mutant L439C(NBD1)/Cys-1074(NBD2), the N-half and C-half proteolytic fragments were present only after treatment with 10 mM DTT. The identity of the N-half and C-half fragments were determined using polyclonal antibody specific for the N-half, and monoclonal antibody A52 that reacts with the A52 epitope tag at the COOH-end of P-gp (data not shown).

Mutant L439C(NBD1)/Cys-1074(NBD2) still retained 82% of the activity of Cys-less P-gp, and cross-linking could be reversed by DTT (Fig. 2C). Therefore, it was of interest to determine whether cross-linking between the nucleotide-binding sites would stimulate or inhibit drug-stimulated ATPase activity. Membranes prepared from HEK 293 cells expressing histidine-tagged Cys-less P-gp or mutant L439C(NBD1)/Cys-1074(NBD2) were treated with or without 0.1 mM Cu²⁺(phenanthroline)₃ for 10 min at 37 °C. The reaction was stopped by addition of EDTA (final concentration 2 mM), and the membranes were diluted with TBS and recovered by centrifugation. The histidine-tagged P-gps were recovered by nickel-chelate chromatography. Fig. 3A shows that treatment of membranes with oxidant did not affect recovery of histidine-tagged P-gp. It appears that the histidine tag remained accessible after cross-linking. Equivalent amounts of purified P-gps were mixed with lipid, sonicated, and assayed for verapamil-stimulated ATPase activity in the presence or absence of DTT. Fig. 3B shows that the activity of Cys-less P-gp was not greatly affected by treatment with oxidant or by the presence of DTT during the ATPase assay. In contrast, the activity of mutant L439C(NBD1)/Cys-1074(NBD2) was reduced by more than 90% after oxidative cross-linking. About 65% of the activity, however, was recovered in the presence of 10 mM DTT. This result indicates that disulfide cross-linking occurred in the active form of P-gp, and cross-linking inhibits ATPase activity.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effect of oxidative cross-linking on ATPase activity. A, histidine-tagged Cys-less (C-less) and mutant L439C(NBD1)/Cys-1074(NBD2) P-gp were isolated by nickel-chelate chromatography after treatment with (+) or without () 0.1 mM oxidant for 10 min at 37 °C. Equivalent amounts of samples were subjected to immunoblot analysis. The positions of cross-linked (X-link) and mature (170-kDa) P-gp are indicated. B, equivalent amounts of purified histidine-tagged Cys-less or mutant L439C(NBD1)/Cys-1074(NBD2) P-gps from oxidant-treated or mock-treated (No oxidant) membranes were mixed with lipid and sonicated with (Oxidant then DTT) or without 10 mM DTT (Oxidant). The samples were then assayed for verapamil-stimulated ATPase activity. The activities are expressed relative to that of a sample that was mock-treated with oxidant and is the average of two different experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The crystal structure of the ATP-binding subunit (HisP) of histidine permease (an ABC transporter) of Salmonella typhimurium was recently reported (15). Histidine permease differs from P-gp in that the permease is a complex of four separate polypeptides; two transmembrane subunits, HisQ and HisM, and two copies of HisP, the ATP-binding subunit. The HisP subunits crystallize as homodimers in the presence of ATP. The phosphate groups of ATP are close to the P-loop, whereas the adenine ring is close to an -helix that is on the COOH-terminal side of the P-loop. The ATP-binding sites face away from each other.

We have attempted to use the cross-linking results to develop a model for the arrangement of the NBDs of P-gp. Fig. 4A shows the model of the arrangement of the NBDs based on the structure of HisP, where the ATP-binding sites face away from each other. Our results, however, favor the model shown in Fig. 4B, in which the ATP-binding sites face each other. A cysteine in the P-loop of NBD1 (G427C) or in the predicted -helix loop that immediately follows the P-loop (L439C) could form a disulfide bond with Cys-1074 in NBD2. The results indicate that at 37 °C, cysteine at positions 427 or 439 can be within 1 Å of Cys-1074. The observation that cross-linking occurred only at 37 °C suggest that some motion or flexibility exists in this region of the NBD.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Models of the arrangement of the NBDs of P-gp. The outward facing (A) and inward facing (B) models are shown. The four domains of P-gp are indicated. These are the N- and C-terminal NBDs (NBD1 and NBD2, respectively) and the N- and C-terminal transmembrane domains (TMD1 and TMD2, respectively). The bold line connected to the cylinder represents the P-loop and -helix structures, respectively, that are predicted to form part of the ATP-binding sites. The endogenous cysteines (Cys-431 and Cys-1074) and the cysteines involved in disulfide bond formation (G427C and L439C) are shown.

It is not known why the orientations of the NBDs of HisP and P-gp are so different. It is possible that the NBDs have a considerable range of motion. This may explain why cross-linking was observed only at the higher temperature. It may also be that the structures of prokaryotic and eukaryotic ABC transporters are indeed different or that the crystal structure is a snapshot of the "resting" phase of the reaction cycle. A combination of crystal structure information and studies such as cross-linking analysis will provide much insight into the mechanism of ABC transporters.

	ACKNOWLEDGEMENTS

We thank Dr. David H. MacLennan for the epitope and monoclonal antibody A52 and Dr. Randal Kaufman for pMT 21. We thank Claire Bartlett for assistance with tissue culture.

	FOOTNOTES

^* This research was supported by National Institutes of Health Grant RO1 CA80900 and grants from the Medical Research Council of Canada 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.

Medical Research Council Scientist and Canadian Cystic Fibrosis Foundation Zellers' Senior Scientist. 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./Fax: 416-978-1105; E-mail: david.clarke@utoronto.ca.

Published, JBC Papers in Press, May 9, 2000, DOI 10.1074/jbc.C000222200

	ABBREVIATIONS

The abbreviations used are: P-gp, P-glycoprotein; DTT, dithiothreitol; NBD, nucleotide-binding domain; ABC, ATP-binding cassette; TBS, Tris-buffered saline; NTA, nitrilotriacetic acid; MalNEt, N-ethylmaleimide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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