From the
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.
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).
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.
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
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.).
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
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.
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.
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
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.
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.).
-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.
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
Mg-ATPase Activity
Using a Colorimetric Assay
-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 NH
Cl, 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.
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 NH
Cl, 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.
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.
Table:
Amino acid analysis was carried out as described
under ``Methods'' on fusion protein C-MBP
-D-galactopyranoside; PAGE,
polyacrylamide gel electrophoresis; CFTR, cystic fibrosis transmembrane
conductance regulator; FPLC, fast protein liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.