©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning, Overexpression, Purification, and Characterization of the Carboxyl-terminal Nucleotide Binding Domain of P-glycoprotein (*)

Sadhana Sharma (§) , David R. Rose

From the (1) Division of Molecular and Structural Biology, Ontario Cancer Institute, and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M4X 1K9 Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Multidrug-resistant tumor cells overexpress P-glycoprotein (170 kDa), a member of the ABC (ATP Binding Cassette)-transporter superfamily. P-glycoprotein has been implicated in transport of a broad range of amphiphilic, hydrophobic drugs from tumor cells. The sequence and structural organization of P-glycoprotein, which consists of 12 transmembrane helices and two cytoplasmic nucleotide binding domains, is similar to other ABC-transporters. It is believed that the nucleotide binding domains of various ABC transporters, which have 30-50% sequence identity, play an important role in coupling ATP hydrolysis to the transport process. To allow structure-function studies of the nucleotide binding domains, the carboxyl-terminal nucleotide binding domain (NBD) of Chinese hamster P-glycoprotein has been cloned, overexpressed, and purified both by itself and as a fusion with maltose-binding protein. It has been demonstrated that the carboxyl-terminal NBD, when overexpressed in Escherichia coli in the absence of transmembrane helices, has very low ATPase activity. This suggests that the amino-terminal nucleotide binding domain and possibly interaction with the transmembrane domains may be required for full ATPase activity. It is also consistent with the idea that the ATPase activity of P-glycoprotein is stimulated in the presence of drugs. Circular dichroism spectral analysis and the ability of carboxyl-terminal NBD, both by itself and as a fusion with maltose-binding protein, to bind ATP-agarose beads and P-glycoprotein specific monoclonal antibodies suggests that the polypeptide folds into a functional domain. Gel filtration chromatography and cross-linking studies indicate that the carboxyl-terminal NBD has a tendency to self-associate to form oligomers. It is speculated that the carboxyl-terminal NBD may play a role in self-association of P-glycoprotein molecules in the plasma membrane.


INTRODUCTION

Multidrug resistance, in which cancer cells manifest resistance not only to chemotherapeutic drugs but also to other structurally unrelated drugs, is a major clinical problem in cancer treatment (for reviews, see Refs. 1 and 2). This phenomenon is associated with the overexpression of the 170-kDa membrane-associated P-glycoprotein (Pgp).() This protein consists of two halves each containing six predicted hydrophobic membrane spanning segments followed by a cytoplasmic domain (3) . The sequence and domain organization of Pgp is typical of the ABC (ATP binding cassette) superfamily of active transporters and is presumably the result of an early gene duplication (4) . The two cytoplasmic domains of Pgp, which include the familiar Walker A and B motifs for nucleotide binding (5) , show extensive sequence identity with the NBDs of other ABC-transporters (6, 7) . Pgp functions as an energy-dependent drug efflux pump and thus plays a major role in the reduced accumulation of drugs inside the cell (8) . Plasma membrane vesicles prepared from multidrug-resistant cells and reconstitution of purified Pgp in vesicles confirms that the drug transport is dependent on the constant supply of energy either from ATP or GTP (9, 10, 11, 12) . Pgp has also been associated with chloride (13) and ATP channel activities (14) , and has peptide transport ability (1) . ATP has been implicated in all the functions of Pgp.

Several residues have been mutated in the Walker A motif of both NBDs of Pgp with varied effects. Some mutations resulted in loss of transport activity but had no effect on binding to ATP analogues, whereas other mutations affected both transport and ATP binding (15) . Also, a double mutation which alters both NBDs simultaneously shows reduced ATP channel activity (14) . However, the mutations which alter the ATP hydrolyzing properties of Pgp have no effect on chloride channel activity. This suggests that unlike drug transport, the chloride channel function of Pgp does not require energy from ATP hydrolysis, although ATP binding is required for channel activation (13).

Several monoclonal antibodies recognizing either the extracellular or cytoplasmic domains of Pgp have been selected (1) . Monoclonal antibody C219 is specific for a continuous short sequence present in both NBDs. Another monoclonal antibody, C494, recognizes only the carboxyl-terminal NBD. The binding of either C219 or C494 to Pgp inhibits the binding of ATP (16) . Another antibody, JC66, which is specific for the distal carboxyl-terminal half of Pgp, completely inhibits the ATP channel activity of Pgp (14) . The fact that C219 and C494 are equally effective in inhibiting ATP binding to Pgp and the inhibition of ATP channel activity by JC66 suggests that there is cooperativity between the NBDs.

The role of the NBDs in Pgp function, the structural changes upon ATP binding, and the coupling of ATP hydrolysis to the transport process remain poorly understood. This is primarily due to the complex structural organization and low abundance of the ABC-transporters (17, 18) . Towards detailed structure-function study of the NBDs, we have cloned, expressed, purified, and characterized the carboxyl-terminal NBD of Chinese hamster Pgp. Three different constructs were made in three different expression systems in an effort to optimize the yield and solubility of the purified protein. Expression as a fusion with maltose-binding protein (C-MBP) resulted in good protein yield, with the advantage that the known MBP structure (19) can aid in crystallographic structure determination of the carboxyl-terminal NBD of Pgp. Circular dichroism, and the ability of the expressed domains to bind ATP and antibodies, suggested that they retain a native-like fold. The fusion protein, C-MBP, retains relatively weak ATPase activity in the absence of transmembrane domains and the other NBD. The tendency of the carboxyl-terminal NBD to form oligomers suggests it may play an important role in self-association of Pgp in the plasma membrane.


EXPERIMENTAL PROCEDURES

Materials

A cDNA clone for Chinese hamster ovary Pgp was obtained from Dr. Victor Ling, Ontario Cancer Institute. Primers used for the polymerase chain reaction (PCR) were synthesized in the Ontario Cancer Institute Biotechnology Laboratory using an Applied Biosystems 392 DNA/RNA synthesizer. The pMal-c2 expression vector, amylose affinity resin, restriction enzymes, Vent DNA polymerase, and T4 DNA ligase were purchased from New England Biolabs. The pT7-7 expression vector was kindly given to us by Dr. Bruce Waygood, University of Saskatchewan. The pET-22b(+) expression vector was obtained from Novagen. Ni-NTA-agarose and DNA purification kit were obtained from Qiagen Inc. ATP-agarose beads were purchased from Sigma. The DNA sequencing kit (Sequenase version 2.0 DNA) was from U. S. Biochemical Corp. A Pharmacia FPLC system was used for gel filtration.

Methods

Construction of the Expression Vector Containing Pgp Carboxyl-terminal NBD

Two different clones of the carboxyl-terminal NBD were amplified by PCR (Fig. 1A). CTM-NBD, which includes the last transmembrane (TM) helix, TM12 (M963) to the end of the transcript was cloned into the expression vector pT7-7 under the control of phage T7 RNA polymerase promoter (Fig. 2). The restriction enzyme sites, NdeI in the forward and SnaBI in the reverse PCR primer shown below, were incorporated to facilitate cloning into vector pT7-7 (20) . Another construct, C-NBD, including amino acids Gln-984 to Arg-1276 but lacking TM12, was also amplified by PCR using the primers shown below with BamHI and EcoRI restriction enzyme sites in the forward and reverse primer, respectively. C-NBD was cloned into pET-22b(+), also under the control of strong phage T7 transcription and translation signals (21) . This expression vector contains a pelB leader sequence for the export of the target protein into the periplasm for improved folding and easier purification (22) . Also at the COOH terminus of the protein there are six consecutive histidine residues. This hexahistidine tag facilitates purification through the high affinity of histidine for Ni-NTA resin (Qiagen Inc.), PCR-NdeI: 5`-GGGAACATATGACATTTGAAAATGTTCTATTAGTAT-3`; PCR-SnaBI: 5`-GGGGTACGTACTTTCTTTTTAACTTTGGTTAAATGC-3`; PCR-BamHI: 5`-GGGGGGGATCCGCAGGTCAGTTCATTTGCT-3`; PCR-EcoRI: 5`-GGGGGGAATTCGCGCTTTGCTCCAGCCTGC-3`.


Figure 1: Amplification of the carboxyl-terminal NBD. A, the Pgp is schematically represented with the transmembrane domains boxed and numbered 1-12 and two NBDs represented by A and B to denote Walker sequences. Two clones CTM-NBD and C-NBD were amplified. B, an agarose gel (1.0%) showing the amplified fragments. Lane 1, 100 base pair ladder; Lane 2, CTM-NBD; Lane 3, C-NBD. C, cloning of CTM-NBD in pT7-7. Various clones were picked, plasmid DNA was isolated. DNA was digested with restriction enzymes NdeI and BamHI to confirm the presence of the insert. Lanes 1, 3, and 4 represent various clones screened for the CTM-NBD insert. D, restriction map of C-NBD. E, restriction digest analysis of C-NBD. Lane 1, markers (1057, 770, 612, 564, 495, 393, 345, and 341 bp); Lane 2, BamHI; Lane 3, PstI (1800 bp fragment); Lane 4, NcoI (530- and 330-bp fragments); Lane 5, XhoI (624 bp fragment); Lane 6, BamHI-HindIII (880-bp fragment); Lane 7, same as lane 1.




Figure 2: The schematic representation of cloning of carboxyl-terminal NBD in the expression vector. The cloning into the expression vector pT7-7, pET22b(+), and subcloning into pMAL-c2 is diagrammatically represented. The amplified DNA fragment of CTM-NBD is ligated into NdeI/SmaI site of pT7-7 expression vector under the control of T7 promoter. The StuI/HindIII fragment from pT7-7 vector containing CTM-NBD fragment was ligated into EcoRI site which was blunt ended with Klenow fragment of DNA polymerase and the HindIII site in the multiple cloning site of pMal-c2 under the control of tac promoter. The insertion of the DNA fragment results in the disruptions of lacZ gene. When the plasmid containing the desired DNA fragment is transformed in lac, complementing host white colonies are formed instead of blue in the presence of 5-bromo-4-chloro-3-indoyl -D-galactoside and IPTG.



The polymerase chain reaction was carried out in a 100-µl reaction volume containing 20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH)SO, 2 mM MgSO, 0.1% Triton X-100, 2.5 units of Vent DNA polymerase, 100 pmol of each primer, and 50 ng of template DNA which had been purified using QIAprep-spin plasmid purifications (Qiagen Inc.). The PCR reaction was carried out for 30 cycles and each cycle was performed as follows: denaturation at 92 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. The amplified PCR products, CTM-NBD (1000 bp) and C-NBD (880 bp), were purified using the QIAquick-spin PCR purification kit (Qiagen Inc.) and subsequently digested with appropriate restriction enzymes. The digested product was then subjected to agarose gel electrophoresis and the DNA was extracted using the QIAEX DNA gel extraction protocol (Qiagen Inc.).

The purified PCR product for CTM-NBD was ligated into the NdeI/SmaI restriction sites of expression plasmid pT7-7 and C-NBD was ligated into the BamHI/EcoRI sites of plasmid pET-22b(+). Competent BL21(DE3)pLysS cells were transformed with the ligation mixture and clones were screened using PCR (23) . The procedure is briefly described as follows: a single colony that is 1 mm in diameter was picked with a sterile pipette tip. Before transferring the colony to an Eppendorf tube containing 50 µl of sterile water, the pipette was touched to a YT+ampicillin (100 µg/ml) plate for later use. The tubes were placed in boiling water for 5 min and centrifuged for 1 min. A 10-µl aliquot of the supernatant was used for the PCR reaction using T7-promoter and terminator primer as described above.

To express the NBD as a fusion with maltose-binding protein, a StuI-HindIII fragment (800 bp), including amino acid Leu-1023 to the end of the transcript, was subcloned from pT7-7 CTM-NBD into an EcoRI restriction enzyme site filled with the Klenow fragment of DNA polymerase and the HindIII restriction site of expression plasmid pMal-c2 (24) (Fig. 2). The insertion of the gene into the vector inactivates the -galactosidase -fragment activity of the malE-lacZ fusion and results in a blue to white color change on 5-bromo-4-chloro-3-indoyl -D-galactoside and isopropyl-1-thio--D-galactopyranoside (IPTG) plates when the construct is transformed into an -complementing host such as JM101 or NM522. The ligation mixture was transformed into competent NM522 Escherichia coli cells and grown on YT-agar plates containing ampicillin, 5-bromo-4-chloro-3-indoyl -D-galactoside, and IPTG. The white colonies were screened by restriction digest analysis and subsequently sequenced using the Sanger dideoxy method (25) to confirm the fidelity of the PCR step.

Overexpression and Purification of CTM-NBD, C-NBD, and C-MBP

BL21(DE3)pLysS cells containing the appropriate construct, either CTM-NBD or C-NBD, were grown in 2 YT broth containing 100 µg/ml ampicillin at 30 °C until A was 0.7. The culture was then induced with 1 mM IPTG and harvested 3 h later at 5000 g for 20 min. After washing with 0.85% (w/v) NaCl, cells were resuspended in lysis buffer consisting of 20 mM Tris-HCl, pH 8.0, 10% glycerol, 500 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 mM -mercaptoethanol, 0.1% Nonidet P-40, 10 µg/ml pepstatin, 10 µg/ml leupeptin, and 1% (v/v) aprotinin. The cells were frozen and kept at -20 °C until required. The frozen cells were thawed in a warm water bath and sonicated on ice for 2 5 min at a power setting 10 and 10% duty cycle with a 5-min interval using a Branson Model 450 sonifier. The lysed cells were centrifuged at 20,000 g in a 45 Ti rotor (Beckmann) for 20 min. For C-NBD, supernatant was loaded onto a Ni-NTA-agarose column equilibrated with lysis buffer without protease inhibitors. After extensive washing, the protein was eluted with lysis buffer plus 200 mM imidazole. The fractions containing protein were detected by SDS-PAGE (26) , pooled, and concentrated.

For CTM-NBD, the crude extract was loaded onto a Q-Sepharose (Pharmacia) column equilibrated with lysis buffer and the protein was eluted with a 0-1 M NaCl gradient. The fractions containing protein were concentrated and loaded onto a S-200HR (Pharmacia) gel filtration column also equilibrated with lysis buffer. After SDS-PAGE analysis, the fractions containing the protein of interest were pooled and concentrated.

For C-MBP, cells were grown, induced, harvested, and lysed in a similar fashion to those for CTM-NBD or C-NBD. The crude extract was loaded onto an amylose-affinity column equilibrated with lysis buffer. The column was extensively washed and the protein was eluted with the same buffer containing 10 mM maltose (27) . Fractions were analyzed by SDS-PAGE, pooled, and dialyzed against 10 mM Tris-HCl, pH 8.0. The sample was then loaded onto a Q-Sepharose column equilibrated with the same buffer. Protein was eluted with a 0-1 M salt gradient in 10 mM Tris-HCl, pH 8.0. Appropriate fractions were pooled, concentrated, and the protein was exchanged into water using an Amicon ultrafiltration cell, with a YM10 membrane (Amicon Inc.).

Amino Acid Analysis of C-MBP

For amino acid analysis, 1000 pmol of C-MBP was vapor-phase hydrolyzed in 6 N HCl for 48 h. The resulting amino acids were derivatized with phenyl isothiocynate and separated on a PICO-TAG (Pharmacia) column (3.8 mm 15 cm) using reverse phase chromatography. The results were analyzed using PICO-TAG amino acid analysis software.

Western Blot Analysis

Western blotting was performed essentially according to Towbin et al. (28) . Briefly, either crude extract or 20-100 µg of purified CTM-NBD, C-NBD, or C-MBP was mixed with an equal volume of gel loading buffer which lacked -mercaptoethanol and electrophoresed on 10% SDS-PAGE. The protein was transferred to a polyvinylidene difluoride membrane using a Bio-Rad electroblotting apparatus. Polyvinylidene difluoride membranes were blocked for 2 h with 10% (w/v) non-fat dry milk in TBS buffer (0.1 M Tris-HCl, pH 7.2, 9% (w/v) NaCl) and then incubated for 1 h with 0.5 µg/ml monoclonal antibody C219 or C494 in the same buffer. The blot was thoroughly washed with TBS containing 1% (w/v) non-fat dry milk and 0.1% (v/v) Tween 20 and then incubated for 1 h with 1:2500 dilution of anti-mouse horseradish peroxidase-conjugated IgG in TBS buffer. After several washes with TBS buffer, the signal was detected by enhanced chemiluminescence (Amersham). The blot was exposed to Kodak X-Omat film for 15 s to 20 min.

ATP-affinity Column Binding

ATP-agarose beads (20 µl) were washed thoroughly with 10 mM Tris-HCl buffer, pH 8.0. Partially purified C-NBD or C-MBP was added to the beads and then incubated on a shaker for 1 h at 4 °C. Agarose beads were washed again with Tris-HCl buffer and then boiled in 30 µl of sample buffer to release protein bound to the beads. The samples were electrophoresed on 12% SDS-PAGE. ATP (1-3 mM) was used to compete the binding of protein to the agarose beads.

Circular Dichroism Spectroscopy (CD)

The fusion protein C-MBP (10 µM) was exchanged into water using an Amicon ultrafiltration cell with a YM30 membrane (Amicon Inc.). The CD spectrum for C-MBP was recorded between 200 and 250 nm at 23 °C in a 1-mm path cuvette using a Jasco J720 spectropolarimeter.

Gel Filtration FPLC

C-MBP (1 mg/ml) in 10 mM Tris-HCl, pH 8.0, was loaded onto a Pharmacia Superose 12 and Superose 6 FPLC gel filtration column equilibrated with the same buffer. The elution profiles of the C-MBP and standards, which consisted of blue dextran (200 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen A (25 kDa) were monitored at 280 nm. The elution volume was calculated and plotted versus log molecular weight. Gel filtration chromatography was also performed in the presence of 0.1% CHAPS in 10 mM Tris-HCl, pH 8.0.

Chemical Cross-linking

Chemical cross-linking of C-MBP was performed with dithiobis(succinimidyl propionate) (DSP) and 3,3`-dithiobis(sulfosuccinimidyl propionate) (DTSSP) (29) . DSP was dissolved in N,N-dimethylformamide to give a final concentration of 2.5 mM and DTSSP was dissolved in 20 mM sodium phosphate buffer, pH 8.0, to a concentration of 0.116 mM. C-MBP (0.029 mM in 20 mM sodium phosphate, pH 8.0) was cross-linked with 0.0135-1.0 mM DSP and 0.0145-0.116 mM DTSSP in 20 mM sodium phosphate buffer. After 20 min at room temperature, the reactions were terminated by adding 50 mM glycine. The samples were loaded on 10% SDS-PAGE after the addition of an equal volume of gel-loading buffer which lacked -mercaptoethanol.

Measurement of Mg-ATPase Activity Using a Colorimetric Assay

Mg-ATPase activity was determined by measuring the release of inorganic phosphate using a colorimetric assay (30) . The assay solution contained 100 µg of C-MBP in 360 µl of assay buffer (50 mM Tris-HCl, pH 7.6, 0.15 mM NHCl, 5 mM MgCl, 2 mM ouabain, 100 µM EGTA, 2 mM dithiothreitol, and 0.89 mM CHAPS). The reaction was initiated by adding 40 µl of ATP to give a final concentration of 2 mM in a 400-µl final volume. After 1 h at 37 °C, the reaction was terminated by adding 400 µl of 6% SDS, 3% ascorbate, and 0.5 M sodium molybdate in 0.5 M HCl. Products were stabilized by adding 400 µl of 2% sodium citrate, 2% sodium meta-arsenite, and 2% acetic acid. After incubation at 37 °C for 10 min the absorbance was read at 850 nm.


RESULTS

Overexpression and Purification of CTM-NBD, C-NBD, and Fusion Protein C-MBP

The amplified PCR products for CTM-NBD and C-NBD (Fig. 1B) were cloned into pT7-7 (Fig. 1C) and pET-22b(+) expression vectors under the control of a strong bacteriophage T7 promoter and the StuI-HindIII fragment from CTM-NBD was subcloned in-frame with maltose-binding protein in the pMal-c2 expression vector (Fig. 2). The correct orientation of the clones was verified by sequencing and restriction digest analysis (Fig. 1, D and E). The IPTG-inducible expression of CTM-NBD and C-NBD and the amount of protein in the soluble fraction were moderate (Fig. 3A). Also the protein repeatedly precipitated during purification. The overall yield of purified protein was 25-50 µg/liter of cell culture. However, when expressed as a fusion with MBP, there was a significant increase in expression level and most of the protein remained soluble (Fig. 3C). Using amylose resin, for which MBP has great affinity, 80 mg of fusion protein was purified from a liter of cell culture. Purified CTM-NBD, C-NBD, and the fusion protein C-MBP were >90% pure as judged by SDS-PAGE (Fig. 3, B and D). The number of amino acids determined by amino acid analysis of C-MBP agreed closely with the theoretical value calculated using the program GCG (Genetics Computer Group, Wisconsin), thus confirming the identity and purity of the fusion protein ().


Figure 3: The SDS-PAGE gels for the analysis of overexpressed and purified protein. BL21(DE3)pLysS cells containing either CTM-NBD or C-NBD were grown and induced as described under ``Methods.'' A, the expression and the localization of CTM-NBD was checked in the various fractions (23). The expression of CTM-NBD appears to be low as shown in the total cell protein of induced cells and seems to be distributed equally in the soluble and insoluble fraction. There was no protein detected in the periplasmic fraction. B, the purified proteins, CTM-NBD (34 kDa) and C-NBD (34 kDa) are shown. C, the expression of C-MBP fusion protein. The total cell protein of uninduced and induced cells is compared. A protein band corresponding to 68 kDa is induced in the presence of 1 mM IPTG, which correspond to C-MBP. The expression of MBP was used as a control and gel shows total cell protein before and after induction. D, the cleavage by specific protease Factor Xa. Factor Xa (200 µg/ml) was added to C-MBP (1 mg/ml) in 50 mM Tris-HCl buffer, pH 8.0, containing 500 mM NaCl and the reaction was carried at room temperature for various lengths of time. Lanes 1 and 2 represent C-MBP before and after 24 h of digestion with Factor Xa.



Expression vector pMal-c2 contains the sequence coding for the recognition site of the specific protease Factor Xa, located 3` of MBP, allowing cleavage of MBP from the protein of interest. Cleavage with Factor Xa for C-MBP was very slow: after 24 h only 20% of the protein was cleaved, suggesting that the protease site may have low accessibility (Fig. 3D).

Western Blot Analysis

The purified proteins were checked for their ability to bind to Pgp-specific monoclonal antibodies, C219 (Fig. 4A) and C494 (Fig. 4, B and C), by Western blot analysis. C219 and C494 recognize all three carboxyl-terminal NBD clones, i.e. CTM-NBD, C-NBD, and C-MBP, expressed in E. coli. MBP alone has no affinity for the antibodies (data not shown). Binding of C-MBP to C219 and C494 was also confirmed by enzyme-linked immunoassay. These results further confirm that the purified protein is the NBD of Pgp and not any other protein from E. coli of the same molecular mass.


Figure 4: Detection of antibody binding by Western blot. Western blot was performed as described under ``Methods.'' The oligomeric forms corresponding to dimeric and trimeric forms of the carboxyl-terminal NBD can be detected by monoclonal antibody. A, detection of monoclonal antibody C219 binding to proteins expressed in E. coli. Lane 1, purified CTM-NBD (50 µg); Lane 2, purified C-NBD (50 µg); Lane 3, purified C-MBP (20 µg). B, binding of monoclonal antibody C494 to carboxyl-terminal NBD. Lane 1, purified CTM-NBD (20 µg); Lane 2, purified C-NBD (10 µg). C, detection of C-MBP (50 µg) with C494.



ATP-agarose Binding Characteristics

CTM-NBD and C-MBP both bind ATP-agarose beads suggesting that the NBD retains its secondary and tertiary structure (Fig. 5A). In a control experiment, MBP alone did not bind the beads indicating that the interactions with ATP are due to the NBD alone. The binding properties of C-MBP, C-NBD, and CTM-NBD were the same. All showed decreased binding to ATP-agarose beads in the presence of ATP (Fig. 5B). CTM-NBD and C-MBP bind to an Affi-Gel Blue affinity gel column (Bio-Rad) (results not shown), which is specific for nucleotide-binding proteins. This further indicates a correctly folded protein that retains ATP binding ability.


Figure 5: ATP binding characteristics of the expressed domains. Partially purified proteins, C-MBP and C-NBD were incubated with agarose beads as described under ``Methods.'' A: Lane 1, C-MBP (partially purified); Lane 2, C-MBP bound to the beads; Lane 3, wash; Lane 4, C-MBP (purified); Lane 5, C-MBP bound to the beads; Lane 6, molecular weight markers; Lane 7, C-NBD (partially purified); Lane 8, C-NBD bound to the beads; Lane 9, wash. B, binding competition with ATP. ATP (1-3 mM) was added to ATP-agarose beads prior to the incubation with C-MBP. Lanes 2, 4, 6, and 8 represent C-MBP bound to the beads in the presence of 0, 1, 2, and 3 mM ATP. Lanes 3, 5, 7, and 9 represent unbound protein.



Secondary Structure of C-MBP as Determined by CD

The ability of the carboxyl-terminal NBD to bind to ATP suggests that it retains a functional tertiary structure. This was further confirmed by CD spectral analysis. A CD spectrum was recorded for C-MBP between 200 and 250 nm. The plot of mean residue molar ellipticity of C-MBP exhibits two minima at 209 and 220 nm (Fig. 6). From the CD spectrum, C-MBP was calculated to consist of 14% -helix, 42% -strand, 20% turn, and 23% irregular structure. This secondary structure calculation was carried out using Prosec software package. Thus, C-MBP is highly structured.


Figure 6: Circular dichroism spectra of C-MBP. The fusion protein C-MBP (10 µM) and MBP (10 µM) was exchanged in water and the CD spectra was recorded as described under ``Methods.'' The plot of molar ellipticity () versus wavelength (nm) is depicted. A, spectra of MBP is shown with dashed line and C-MBP with solid line.B, difference spectrum which represents spectrum of NBD was obtained by subtracting spectra of C-MBP from MBP.



Oligomer Formation

The tendency of the carboxyl-terminal NBD to form oligomers was evident from gel filtration and chemical cross-linking experiments. In the former, most C-MBP elutes at the void volume (with the blue dextran) of an FPLC gel filtration column, exhibiting a molecular mass of 2000 kDa. However, a small fraction of protein also elutes with bovine serum albumin which has about the same M as C-MBP (Fig. 7). Performing gel filtration chromatography in the presence of 0.1% (v/v) CHAPS had no effect on the elution profile of the standards and C-MBP (results not shown). In a control gel filtration experiment involving MBP alone, there was no evidence for oligomerization. The tendency of C-MBP to oligomerize was further evidenced by using the homobifunctional, thio-cleavable and amine-reactive chemical cross-linkers DSP and DTSSP. With such reagents, proteins in close proximity have the potential to be chemically cross-linked. With a low concentration of DTSSP (0.0073, 0.0145, and 0.029 mM), bands which correspond to dimeric (136 kDa) and trimeric (204 kDa) forms of C-MBP are seen (Fig. 8). With DSP, such intermediate forms could not be detected and most protein was present as higher order oligomers. MBP alone did not form oligomers with either DTSSP or DSP. Furthermore, Western blot analysis confirms that the Pgp-specific monoclonal antibodies are able to recognize the oligomeric forms of CTM-NBD, C-NBD, and C-MBP (Fig. 4).


Figure 7: Molecular weight by gel filtration FPLC. The elution volume of the standards which includes blue dextran, bovine serum albumin, ovalbumin, and chymotrypsinogen A was determined and plotted as a function of log molecular weight. Elution volume of purified C-MBP was determined under the same conditions. Molecular mass of C-MBP was estimated to be approximately 2000 and 68 kDa shown by arrows on the graph.




Figure 8: Identification of intermediate oligomeric forms of C-MBP by chemical cross-linking with DSP and DTSSP. A series of cross-linking experiments were performed with purified 0.029 mM C-MBP as described under ``Methods.'' Lanes 1-4 shows cross-linking with increasing concentration of DSP (0.0135, 0.135, 0.5, and 1.0 mM); lane 5 is molecular weight markers; Lanes 7-10 represent cross-linking with increasing concentration of DTSSP (0.0073, 0.015, 0.029, 0.058, and 0.116 mM).



ATPase Activity of the Fusion Protein

C-MBP exhibits very low Mg-dependent ATPase activity with a V of 23.8 nmol/min/mg and a K of 20 mM as determined by a Lineweaver-Burk plot (Fig. 9). The presence of ouabain (2 mM) and EGTA (100 µM) has no effect on the activity, suggesting that the observed ATPase activity is not due to the presence of contaminating Na-K- or Ca-ATPase. ATPase activity is dependent on the concentration of ATP and Mg in the assay buffer. In the presence of 5 mM MgCl, ATPase activity decreased with increasing concentration of ATP. The observed decrease may be due to a limiting concentration of Mg in the assay buffer, since no decrease in activity with increasing ATP concentration was observed when the assay was performed in the presence of 60 mM MgCl. In the reverse experiment, in which ATP concentration was kept constant, a similar dependence of ATPase activity on Mg concentration was observed (Fig. 10): in the presence of 2 mM ATP, ATPase activity decreased as Mg concentration increased. The observed decrease can be attributed to a limiting concentration of ATP. The kinetic parameters of the carboxyl-terminal NBD, in the absence of the Pgp transmembrane helices, are different from those of intact Pgp. The ATPase activity of Chinese hamster Pgp decreases in the presence of high concentrations of ATP with maximal activity at 6 mM ATP (12) . In some cases a higher Mg concentration (50 mM) caused inhibition of ATPase activity and in other cases there was no inhibition up to 50 mM Mg(12) .


Figure 9: Lineweaver-Burk plot of dependence of ATPase activity of fusion protein C-MBP. Assay buffer contained 100 µg of C-MBP in 400 µl of 400 µl of 50 mM Tris-HCl, pH 7.6, 0.15 mM NHCl, 60 mM MgCl, 2 mM ouabain, 100 µM EGTA, 2 mM dithiothreitol, 0.89 mM CHAPS, and various concentrations of ATP (1-10 mM). V and K was calculated from the plot of 1/V (1/nmol of ATP hydrolyzed per min per mg of C-MBP) versus 1/S (1/[ATP] mM). V of 23.8 nmol/min/mg and a K of 20 mM for ATP was calculated from the plot.




Figure 10: Dependence of ATPase activity on concentrations of MgCl and ATP. Each assay sample contained 100 µg of C-MBP in 400 µl of 50 mM Tris-HCl, pH 7.6, 0.15 mM NHCl, 5 mM MgCl, 2 mM ouabain, 100 µM EGTA, 2 mM dithiothreitol, and 0.89 mM CHAPS or as indicated. A, ATP dependence in the presence of 5 mM MgCl. B, ATP dependence in the presence of 60 mM MgCl. C, Mg dependence in the presence of 3 mM ATP.




DISCUSSION

A central question in multidrug resistance is how a single integral membrane protein, Pgp, can transport a wide variety of drugs and hydrophobic peptides. The role of ATP and how ATP-derived energy is harnessed in the transport process of Pgp and other ABC-transporters remains unknown. It has been suggested that the transport of solutes by an ABC-transporter across the membrane is dependent on energy derived from ATP and that two molecules of ATP are hydrolyzed per transport event (31) . The latter proposal is consistent with the fact that most ABC-transporters contain two NBDs. The hemolysin and glycine-betaine transporters, which have only one NBD, are known to function as dimers (17). The NBDs from several ABC-transporters, for example, cystic fibrosis transmembrane conductance regulator (CFTR) and maltose (MalK), histidine (HisP), and oligopeptide (OppF) permeases, have been overexpressed and purified (32, 33, 34, 35, 36) . The purified domains are able to bind ATP and its analogues but show no ATPase activity. It has also been suggested that these nucleotide-binding domains may not be able to hydrolyze ATP in the absence of the membrane binding components of the transporters (31) .

In this study, we have chosen the carboxyl-terminal NBD of Pgp as a model system for structural and functional characterization of ABC-transporter NBDs. Early reports suggest that, like any other ABC-transporter, Pgp requires both of its NBDs for efficient activity and also that the ATPase activity is stimulated in the presence of drugs (33) . Georges et al.(1991) suggested that there is cooperativity between the two NBDs of Pgp, as evidenced by the study of mutants having deletions in the amino or carboxyl half of Pgp. Deletion mutations in either domain resulted in the loss of transport function, indicating that both halves are necessary (37, 38) . This contradicts the finding that the amino-terminal NBD alone contains all the residues necessary to hydrolyze ATP (39) . These studies were carried out with either intact Pgp or the isolated amino-terminal half including TMDs 1-6. To investigate the role of the carboxyl-terminal NBD of Pgp in transport, chloride, and ATP channel activities, we have overexpressed this domain in E. coli.

When overexpressed, the CTM-NBD and C-NBD proteins were found in the membrane fraction, indicating that either the protein is very hydrophobic or is associated with the membrane. This is not surprising for CTM-NBD since it contains the amino acid residues from TM12. C-NBD, which lacks TM12 residues, was expressed in pET22b(+). This expression plasmid contains a pelB leader sequence, which directs proteins to the periplasmic space. Since C-NBD also remained associated with the membrane, it might be speculated that in intact Pgp, the NBDs somehow associate with the membrane. Similar observations have been made with the NBDs of bacterial permeases such as HisP, OppF, and MalK and with those of human CFTR. When expressed in the absence of TMDs, these NBDs remain closely associated with the membrane (32, 33, 34, 35, 36) . Although the NBDs and the TMDs of bacterial ABC-transporters are encoded by different sets of genes, the proteins tightly associate with each other to form the intact transporter. It is interesting to speculate how NBDs which are highly hydrophilic might interact with the membrane. It was proposed that a segment of NBD protrudes into or through a pore generated by the TMDs (35) . However, this remains to be verified by, for example, three-dimensional structural information.

Expression of the carboxyl-terminal NBD as a fusion with MBP showed tremendous improvement in the protein yield and solubility. There are additional advantages to expressing protein as a fusion with MBP: the affinity of MBP for amylose can be used for rapid purification and the three-dimensional structure of MBP has been elucidated (19) . This could yield information useful in the ultimate structure determination of the Pgp NBD. The inability to efficiently cleave MBP from the NBD with Factor Xa suggests that the protease site has low accessibility. A similar observation was made with the NBD of CFTR when expressed as a fusion with MBP. It was speculated that the CFTR NBD may interact strongly with MBP (33) .

CTM-NBD and C-MBP retain the ability to bind ATP. Unlike NBDs of other ABC-transporters, the carboxyl-terminal NBD of Pgp retains low ATPase activity when expressed in the absence of the other NBD and TMDs. Previous work suggests that upon mutating one of the two NBDs, Pgp loses its activity, indicating that the two NBDs function in a cooperative manner (37) . Interactions of the carboxyl-terminal NBD with either or both of the TMDs and the amino-terminal NBD of Pgp may be required for full ATPase activity. Inhibition of ATPase activity with a high concentration of both Mg and ATP, as observed with intact Pgp, was not observed for the carboxyl-terminal NBD. This suggests that some regulation of the ATPase activity of intact Pgp is lost when the NBD is expressed in the absence of the TMDs and the other NBD. These results are also consistent with the fact that ATPase activity of intact Pgp is stimulated in the presence of drugs.

The NBDs of Pgp share considerable sequence similarity with the NBDs of CFTR. For example, there is 32% identity and 56% similarity between the carboxyl-terminal NBD of Pgp and CFTR nucleotide binding fold 1. However, the carboxyl-terminal domain of Pgp shows rather weak binding to TNP-ATP, a fluorescent analogue of ATP, unlike nucleotide binding fold 1 of CFTR (33) . Such differences in ATP binding behavior of these domains possibly evidences subtle active-site structural differences.

It has previously been observed that Pgp exists as a dimer or trimer in the plasma membrane (40, 41) . The results from this study, showing that CTM-NBD, C-NBD, and C-MBP tend to form dimers, trimers, and higher oligomers, suggest that the carboxyl-terminal NBD may play an important role in the self-association of Pgp within the plasma membrane. One can speculate from these results that intact Pgp molecules undergo ordered self-association through interactions between NBDs. However, an NBD in the absence of TMDs and the other NBD may undergo less ordered self-association with the resulting tendency to form higher oligomers.

The successful expression and purification of the carboxyl-terminal NBD of Pgp is a critical step in structure-function studies of the molecule. In combination with an analogous approach to the expression and characterization of the amino-terminal NBD, we hope to improve understanding of the role of the NBDs in the function of ABC-transporters.

  
Table: Amino acid analysis was carried out as described under ``Methods'' on fusion protein C-MBP

The molar ratios are based on 98 mol of His/mol of fusion protein. The value for tryptophan is not reported because it gets degraded in presence of 6 M HCl and is not detectable. The low value of cysteine is also attributed to the degradation during hydrolysis.



FOOTNOTES

*
This work was supported by the National Cancer Institute of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 416-924-0671 (ext: 5053); Fax: 416-926-6529; E-mail: ssharma@oci.utoronto.ca.

The abbreviations used are: Pgp, P-glycoprotein; ABC-transporters, ATP binding cassette transporters; NBD, nucleotide binding domain; TM, transmembrane; TMD, transmembrane domain; MBP, maltose-binding protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DSP, dithiobis(succinimidyl propionate); DTSSP, 3,3`-dithiobis(sulfosuccinimidyl propionate); PCR, polymerase chain reaction; bp, base pair(s); IPTG, isopropyl-1-thio--D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; CFTR, cystic fibrosis transmembrane conductance regulator; FPLC, fast protein liquid chromatography.


ACKNOWLEDGEMENTS

Drs. S. Bagby, M. Ikura, and P. Yau are thanked for their help, encouragement, and scientific discussions. S. Bagby's help in preparing and critical reading of the manuscript is greatly appreciated. We acknowledge the help of Dr. A. Shapiro with ATPase assays and Dr. A. Chakrabartty with CD analysis.


REFERENCES
  1. Endicott, J., and Ling, V.(1989) Annu. Rev. Biochem. 58, 137-171 [CrossRef][Medline] [Order article via Infotrieve]
  2. Gottesman, M. M., and Pastan, I.(1993) Annu. Rev. Biochem. 62, 385-427 [CrossRef][Medline] [Order article via Infotrieve]
  3. Chen, C. J., Chin, J. E., Ueda, K., Clark, D. P., Pastan, I., Gottesman, M. M., and Roninson, I. B.(1986) Cell 47, 381-389 [Medline] [Order article via Infotrieve]
  4. Gros, P., Croop, J., and Housman, D. E.(1986) Cell 47, 371-390 [Medline] [Order article via Infotrieve]
  5. Walker, J. E., Sarste, M., Runswick, M. J., and Gay, N. J.(1982) EMBO J. 1, 945-951 [Medline] [Order article via Infotrieve]
  6. Hyde, S. C., Emsley, P., Hartshon, M. J., Mimmack, M. M., Gileadi, U., Pearce, S. R., Gallagher, M. P., Gill, D. R., Hubbard, R. E., and Higgins, C. F.(1990) Nature 346, 362-365 [CrossRef][Medline] [Order article via Infotrieve]
  7. Mimura, C. S., Holbrook, S. R., and Ames, G. F. L.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 84-88 [Abstract]
  8. Kessel, D., Botterill, V., and Wodinsky. I.(1968) Cancer Res. 28, 938-941 [Medline] [Order article via Infotrieve]
  9. Lelong, I. H., Padmanabhan, R., Lovelace, E., Pastan, I., and Gottesman, M. M.(1992) FEBS Lett. 304, 256-260 [CrossRef][Medline] [Order article via Infotrieve]
  10. Al-Shawi, M., and Senior, A. E.(1993) J. Biol. Chem. 268, 4197-4206 [Abstract/Free Full Text]
  11. Sharom, F. J., Xiaohong, Y., and Doige, C. A.(1993) J. Biol. Chem. 268, 24197-24202 [Abstract/Free Full Text]
  12. Shapiro, A. B., and Ling, V.(1994) J. Biol. Chem. 269, 3745-3754 [Abstract/Free Full Text]
  13. Gill, D. R., Hyde, S. C., Higgins, C. F., Valverde, M. A., Mintenig, G. M., and Sepulveda, F. V.(1992) Cell 71, 23-32 [Medline] [Order article via Infotrieve]
  14. Abraham, E. H., Prat, A. G., Gerweck, L., Seneveratne, T., Arceci, R. K., Guidotti, G., and Cantiello, H. F.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 312-316 [Abstract]
  15. Azzaria, M., Schurr, E., and Gros, P.(1989) Mol. Cell. Biol. 9, 5289-5297 [Medline] [Order article via Infotrieve]
  16. Georges, E., Zhang, J.-T., and Ling, V.(1991) J. Cell. Physiol. 148, 479-484 [Medline] [Order article via Infotrieve]
  17. Higgins, C. F., Hyde, S. C., Mimmack, M. M., Gileadi, U., Gill, D. R., and Gallgher, M. P.(1990) J. Bioenerg. Biomembr. 22, 571-592 [Medline] [Order article via Infotrieve]
  18. Higgins, C. F.(1992) Annu. Rev. Cell Biol. 8, 67-113 [CrossRef]
  19. Spurlino, J. C., Lu, G. Y., and Quiocho, F. A.(1991) J. Biol. Chem. 266, 5202-5219 [Abstract/Free Full Text]
  20. Tabor, S., and Richardson, C. C.(1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1074-1078 [Abstract]
  21. Studier, F., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89 [Medline] [Order article via Infotrieve]
  22. Lei, S. P., Lin, H. C., Wang, S. S., Callaway, J., and Wilcox, G. (1987) J. Bacteriol. 169, 4379-4383 [Medline] [Order article via Infotrieve]
  23. Novagen, Inc.(1992) pET System Manual, Novagen Inc. (company newsletter)
  24. Guan, C., Li, P., Riggs, P. D., and Inouye, H.(1987) Gene (Amst.) 67, 21-30 [CrossRef]
  25. Sanger, F., Nicklen, S., and Coulson, A. R.(1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  26. Laemmli, U. K.(1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  27. Maina, C. V., Riggs, P. D., Grandea, A. G., III, Slatko, B. E., Moran, L. S., Tagliamonte, J. A., McReynolds, L. A., and Guan, C.(1988) Gene (Amst.) 74, 365-373 [CrossRef][Medline] [Order article via Infotrieve]
  28. Towbin, H., Staehelin, T., and Gordon, J.(1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  29. Maman, J. D., Yager, T. D., and Allan, J.(1994) Biochemistry 33, 1300-1310 [Medline] [Order article via Infotrieve]
  30. Clifflet, S., Torriglia, A., Chiesa, R., and Tolosa, S.(1988) Anal. Biochem. 168, 1-4 [Medline] [Order article via Infotrieve]
  31. Mimmack, M. L., Gallagher, M. P., Pearce, S. R., Hyde, S. C., Booth, I. R., and Higgins, C. F.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8257-8261 [Abstract]
  32. Hartman, J., Huang, Z., Rado, T. A., Peng, S., Jilling, T., Muccio, D. D., and Sorscher, E. J.(1992) J. Biol. Chem. 267, 6455-6458 [Abstract/Free Full Text]
  33. Ko, Y. H., Thomas, P. J., Delannoy, M. R., and Pederson, P. L.(1993) J. Biol. Chem. 268, 24330-24338 [Abstract/Free Full Text]
  34. Shuman, H. A., and Silhavy, T. J.(1980) J. Biol. Chem. 256, 560-562 [Abstract/Free Full Text]
  35. Kerppola, R. E., Shyamala, V. K., Klebba, P., and Ames, G. F.(1991) J. Biol. Chem. 266, 9857-9865 [Abstract/Free Full Text]
  36. Gallagher, M. P., Pearce, S. R., and Higgins, C. F.(1989) Eur. J. Biochem. 180, 133-141 [Abstract]
  37. Azzaria, M., Schurr, E., and Gros, P.(1989) Mol. Cell. Biol. 9, 5289-5297 [Medline] [Order article via Infotrieve]
  38. Ambudkar, S. V., Lelong, I. H., Zhang, J., Cardarelli, C. O., Gottesman, M. M., and Pastan, I.(1993) Proc. Natl. Acad. Sci. U. S. A. 89, 8472-8476 [Abstract]
  39. Shimabuku, A. M., Nishimoto, T., Ueda, K., and Komano, T.(1992) J. Biol. Chem. 267, 4308-4311 [Abstract/Free Full Text]
  40. Boscoboinik, D., Debanne, M. T., Stafford, A. R., Jung, C. Y., Gupta, R. S., and Epand, R. M.(1990) Biochim. Biophys. Acta 1027, 225-228 [Medline] [Order article via Infotrieve]
  41. Poruchynsky, M. S., and Ling, V.(1994) Biochemistry 33, 4163-4174 [Medline] [Order article via Infotrieve]

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