(Received for publication, November 7, 1995; and in revised form, March 4, 1996)
From the
Varying length cDNAs encoding the N-terminal nucleotide-binding domain (NBD1) from mouse mdr1 P-glycoprotein were prepared on the basis of structure predictions. Corresponding recombinant proteins were overexpressed in Escherichia coli, and the shortest one containing amino acids 395-581 exhibited the highest solubility. Insertion of an N-terminal hexahistidine tag allowed domain purification by nickel-chelate affinity chromatography.
NBD1
efficiently interacted with nucleotides. Fluorescence methods showed
that ATP bound at millimolar concentrations and its
2`,3`-O-(2,4,6-trinitrophenyl) derivative at micromolar
concentrations, while the 2`(3`)-N-methylanthraniloyl
derivative had intermediate affinity. Photoaffinity labeling was
achieved upon irradiation with 8-azido-ATP. The domain exhibited ATPase
activity with a K for MgATP in the
millimolar range, and ATP hydrolysis was competitively inhibited by
micromolar 2`,3`-O-(2,4,6-trinitrophenyl)-ATP.
NBD1
contained a single cysteine residue, at position 430, that was
derivatized with radiolabeled N-ethylmaleimide. Cysteine
modification increased 6-fold the K for
2`(3`)-N-methylanthraniloyl-ATP and prevented 8-azido-ATP
photolabeling. ATPase activity was inhibited with a 5-fold increase in
the K
for MgATP. The results suggest that
chemical modification of Cys-430 is involved in the N-ethylmaleimide inhibition of whole P-glycoprotein by
altering substrate interaction.
Multidrug resistance of tumor cells is often associated with overexpression of P-glycoprotein, a membrane transporter that extrudes chemotherapeutic drugs using ATP hydrolysis as energy source(1, 2) . The protein is encoded by the mdr gene family comprising two members in man, mdr1 and mdr2, or three in mouse, mdr1 (or mdr1b), mdr2, and mdr3 (or mdr1a). Only mdr1, and to a lower extent mdr3, was found to convey cellular multidrug resistance; mdr3 appears to be involved in detoxification/protection processes and mdr2 in phospholipid translocation(3) . The function of mdr1 P-glycoprotein in normal tissues is still questioned although its relative abundance in mouse pregnant uterus and adrenal glands (4) favors a role in steroid hormone secretion(5) .
Structural analysis of the
P-glycoprotein sequence, composed of 1276 amino acids in
mouse(6) , predicts two homologous halves, each containing up
to six putative membrane-spanning -helices and one cytoplasmically
sided nucleotide-binding domain with characteristic Walker motifs A and
B(7) . P-glycoprotein structural organization is typical of the
ATP-binding cassette (ABC) (
)superfamily including yeast (8) and protozoan parasite (9) drug transporters, and a
series of different members from eukaryotic proteins, like the cystic
fibrosis gene product CFTR, to bacterial transporters(10) . The
ATPase activity and related drug transport of P-glycoprotein require
both functional nucleotide-binding sites (11, 12) and
are sensitive to the cysteine-specific modifier N-ethylmaleimide
(NEM)(13, 14, 15, 16, 17) .
The lack of structural data about P-glycoprotein is due to its low
abundance, difficult purification and membrane character, and to the
lack of a highly overexpressing system(18) . A recent approach
to circumvent such problems was to overexpress in bacteria recombinant
domains predicted to be soluble, in fusion with either the glutathione S-transferase or the maltose-binding protein to allow their
purification by affinity chromatography: this was achieved with the
C-terminal nucleotide-binding domain (NBD2) from
P-glycoprotein(19, 20) , or with HlyB or CFTR
domains(21, 22) . However, the presence of a
relatively high-size fusion protein might be undesirable when studying
protein/protein interactions, and its release by specific proteolytic
cleavage was only partial or led to unstable nucleotide-binding
domains. An alternative was to use a hexahistidine tag for fusion, in
order to increase protein solubility and allow its purification by
nickel-chelate chromatography.
The aim of the present work was to design the N-terminal nucleotide-binding domain (NBD1) from mouse P-glycoprotein encoded by the mdr1 gene using structure predictions and to overexpress it in E. coli as a hexahistidine-tagged recombinant protein to study nucleotide interactions and the possible role of its single cysteine residue. The results indicate that NBD1 of varying length was highly overexpressed, but only the shortest one exhibited sufficient solubility to be purified as milligram amounts of protein. The domain efficiently bound ATP or analogues and exhibited ATPase activity; chemical modification of the single cysteine residue by NEM altered nucleotide interaction and substrate hydrolysis.
The primers allowed the
introduction of BamHI and HindIII restriction sites,
and the amplified cDNA of 747 base pairs was digested by endonucleases
and ligated into the corresponding sites of linearized pQE-30 plasmid
(Qiagen). E. coli JM105 cells (supE endA sbcB15 hsdR4 rpsL thi (lac-proAB) F`
[traD36 proAB
lacI
lacZ
M15]) were
transformed with the ligation product and grown on agar plates
supplemented with ampicillin (50 µg/ml).
The recombinant plasmid was used as a template to produce by PCR amplification the cDNAs to be inserted in the pQE-30 plasmid and coding for NBD1-613 and NBD1-581, by using the same Asn-395-related primer together with the following one, 5`-TATAAGCTTCTACTCATCATGATTTCCTTGCTCCACAA-3` or 5`-TATAAGCTTCTAGGTGGTCCGGCCTTCTCTAGCCTTAT-3`, corresponding to C-terminal Glu-613 or Thr-581, respectively. In all cases, the PCR conditions were the following: (i) a first denaturation at 92 °C for 4 min, (ii) 35 cycles each consisting in denaturation at 92 °C for 30 s and elongation at 72 °C for 60 s plus a 1-s increase per cycle, and (iii) a final elongation at 72 °C for 10 min.
The three recombinant plasmids were restriction-mapped, and the dideoxy sequencing method confirmed the expected sequences for NBD1 cDNAs by reference to the published cDNA sequence of mdr1 P-glycoprotein(6) .
The supernatant was applied to a
Ni-nitrilotriacetic acid column (24) equilibrated in 50 mM potassium phosphate, 150
mM Na
SO
, 1% (v/v) Triton X-100, 10%
(w/v) glycerol, 40 mM imidazole, at pH 8.5. The column was
first extensively washed with the same buffer containing 0.7 M NaCl, and then in the absence of Triton X-100 and NaCl but in the
presence of 0.05% (w/v) HECAMEG. The retained proteins were then eluted
with 200 mM imidazole, in the presence of 0.01% HECAMEG, and
analyzed by SDS-polyacrylamide gel electrophoresis. The fractions were
pooled and dialyzed against 50 mM potassium phosphate, 150
mM Na
SO
, 20% glycerol, 0.01% HECAMEG
(dialysis buffer) at pH 8.5. The dialysate was centrifuged to discard
possible traces of precipitated material; the supernatant
(0.2-0.3 mg of protein/ml) was aliquoted and kept frozen in
liquid nitrogen.
Protein fractions were analyzed on 12% SDS-polyacrylamide gels as described by Laemmli(25) . Protein concentration was routinely determined by the method of Bradford (26) with the Coomassie Blue Plus Protein Assay Reagent kit from Pierce.
Fluorescence measurements were performed by diluting protein solutions (3-5 µM final concentration) in 1 ml of dialysis buffer, at pH 8.5, in the presence of increasing concentrations of fluorescent ATP analogues. When the extrinsic fluorescence of TNP-ATP was studied, the excitation was performed at 408 nm and emission was scanned in the range 540-570 nm. For MANT-ATP, the excitation wavelength was 350 nm and emission was scanned from 400 to 480 nm. ATP analogue binding was determined from the increase in fluorescence at 545 nm or 432 nm, respectively, for TNP-ATP or MANT-ATP, in the presence as compared to the absence of NBD1-581. Curve-fitting of the concentration-dependent analogue binding was performed with the Grafit program (Erithacus software) as detailed previously(28, 29) . For ATP-dependent chase of bound TNP-ATP, controls were conducted with the same nucleotide concentrations but in the absence of protein, and curve-fitting was analyzed according to Stinson and Holbrook(30) .
Tyrosine-intrinsic fluorescence of 1 µM NBD1-581 (5 µM tyrosine residues) was measured upon excitation at 275-280 nm by scanning emission in the range 285-360 nm. The binding of MANT-ATP monitored by quenching of emission fluorescence was studied in the presence of increasing analogue concentrations. Correction for nucleotide inner filter effect was determined under the same conditions using N-acetyltyrosinamide. Curve-fitting of nucleotide analogue binding was performed with the Grafit program as described previously for quenching of tryptophan-intrinsic fluorescence(19, 28, 29) .
Figure 1: Schematic structural organization of mouse membrane-inserted P-glycoprotein and recombinant N-terminal nucleotide-binding domain. Top, topological model of P-glycoprotein showing the putative transmembrane helices (dashed areas), the two cytoplasmic nucleotide-binding domains, NBD1 and NBD2, each containing the characteristic Walker motifs A and B of ATP site, the ABC-transporter signature (S) and a C219 monoclonal-antibody epitope, and finally the central, phosphorylatable, linker region. Bottom, recombinant hexahistidine-tagged NBD1 of variable size with respect to the C-terminal side (NBD1-581, NBD1-613, or NBD1-643) and containing a single cysteine residue inside the Walker motif A.
Fig. 2shows the analysis of the NBD1-containing 350-708
amino acid sequence from mouse mdr1 P-glycoprotein by using
the ANTHEPROT program(34) . Four different predictive methods
were used concerning hydrophobicity(35) ,
accessibility(36) , antigenicity(37) , and secondary
structures(38) . Domain design answered the following criteria:
(i) the minimal size had to contain the ABC-transporter signature S and
both Walker motifs A and B whose critical roles were shown in other
ATP-binding proteins of known three-dimensional structure like
adenylate kinase, RecA, or mitochondrial F-ATPase; (ii) the
maximal size was limited by membrane proximity on the N-terminal side
and by the phosphorylatable linker region starting around position 660 (39) on the C-terminal side; (iii) the limits had to be located
inside hydrophilic, accessible, antigenic, and aperiodic regions. Due
to the reverse transcriptase-PCR method used for cDNA cloning, an
additional constraint was to find mdr1 product sequences
sufficiently different from the mdr2 and mdr3 ones,
such sequences being rare due to the high similarity in primary
structure of the three proteins(40) . To fulfill all these
conditions, Asn-395 and Ser-643 were chosen, respectively, as
N-terminal and C-terminal ends for NBD1 (named NBD1-643); the
corresponding cDNA was prepared by reverse transcriptase-PCR from
adrenal cells which contain abundant mdr1 mRNA, limited
amounts of mdr3 mRNA, and almost no mdr2
mRNA(4) . Two other C-terminal limits, Glu-613 and Thr-581
giving the respective shorter domains NBD1-613 and
NBD1-581, were determined. Whereas the Thr-581 environment
appears hydrophobic inside whole P-glycoprotein, due to following
apolar residues, it acquires a hydrophilic value when the residue
occupies the C-terminal position. The corresponding cDNAs were prepared
by PCR amplification using the cDNA coding for NBD1-643 as a
template, and a N-terminal hexahistidine tag was linked to all
recombinant domains (cf.Fig. 1).
Figure 2: Determination of NBD1 limits from structure predictions. The 350-708-amino acid sequence from mouse P-glycoprotein encoded by the mdr1 gene was analyzed with the ANTHEPROT program using predictions of hydrophobicity (a), accessibility (b), antigenicity (c), and secondary structures (d). At the bottom of the figure are positioned the Walker motifs A and B, the ABC transporter signature (S), the C219 monoclonal-antibody epitope, and the phosphorylatable linker region. The differences in sequence of N-terminal and C-terminal ends of the mdr1 product, around positions 395 and 643, with respect to the mdr2 and mdr3 ones are also shown, whereas the points represent conserved residues. The chosen limits are indicated with dashed vertical lines.
Figure 3:
Differential overexpression of the cDNA
coding for NBD1 of varying length. Fractions from 5-ml cultures of E. coli cells overexpressing the cDNA coding for
NBD1-643 (A), NBD1-613 (B), or
NBD1-581 (C) were analyzed by SDS-polyacrylamide gel
electrophoresis. Lane 1, total bacteria proteins before IPTG
induction (loaded sample equivalent to 50 µl of culture); lane
2, total bacteria proteins after IPTG induction (sample equivalent
to 30 µl of culture); lane 3, bacteria lysate after
sonication; lane 4, insoluble fraction recovered in the pellet
of centrifugation; lane 5, soluble proteins from the
supernatant. The scale on the right of each panel
indicates the positions of molecular mass markers run under the same
conditions (cf.Fig. 4) and corresponding, from top to bottom, to phosphorylase b (94 kDa), bovine
serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (29
kDa), soybean trypsin inhibitor (20 kDa), and -lactalbumin (14.4
kDa). The tip of each arrow indicates the position of
the corresponding recombinant domain.
Figure 4: Preparative overproduction and purification of the NBD1-581 domain. Overexpression was performed under conditions of Fig. 3C using a 3-liter culture. Lanes 1 and 2, as in Fig. 3; lane 3, soluble fraction after French Press treatment; lane 4, fraction not retained on the nickel-chelate resin; lane 5, purified domain retained on the column and eluted with 200 mM imidazole; its position is indicated by the arrow on the right. The molecular mass markers (MW), with indicated values on the left, are described in the legend of Fig. 3.
A preparative
purification of NBD1-581 is illustrated in Fig. 4.
Similarly as in Fig. 3C, the overexpressed domain
appeared abundant in both the total protein fraction (lane 2)
and the soluble one (lane 3). Affinity chromatography using a
nickel-chelate resin was particularly efficient to discard bacterial
proteins as a pass-through fraction (lane 4) and to
selectively bind and purify the NBD1-581 domain which was then
eluted with 200 mM imidazole (lane 5). After dialysis
in 50 mM potassium phosphate, 150 mM NaSO
, 20% glycerol, 0.01% HECAMEG, at pH
8.5, about 4 mg of protein of approximately 95% pure NBD1-581, at
a 10-15 µM concentration, could be obtained from a
3-liter culture. Purified NBD1-581 reacted strongly with C219
monoclonal antibody, which is specific for the VQXALD
sequence(42) , when assayed by immunoblotting (not shown here).
The binding of MANT-ATP,
another fluorescent derivative, was also monitored by the enhancement
of extrinsic fluorescence as illustrated in Fig. 5A.
When MANT-ATP bound to NBD1-581, its fluorescence intensity
increased while the wavelength of maximal emission was blue-shifted
from 444 to 432 nm. At fixed protein concentrations, the increase at
432 nm was dependent on MANT-ATP concentration in a saturable manner
and allowed the determination of a K value for
MANT-ATP of 25.2 ± 4 µM (Fig. 5B).
The binding of MANT-ATP could also be monitored by changes in the
intrinsic fluorescence of NBD1-581 which contained five tyrosine
residues. Fig. 5C illustrates the
tyrosine-characteristic fluorescence of 1 µM NBD1-581: the excitation spectrum exhibited a maximum at 275
nm, with a shoulder around 280 nm, whereas the emission was maximal at
299 nm. Addition of MANT-ATP produced a significant quenching of the
domain fluorescence emission. The quenching was dependent on the
analogue concentration (Fig. 6, empty symbols) giving a K
value for MANT-ATP of 25 ± 8
µM. Incubation of NBD1-581 for 1 h with increasing
concentrations of radioactive NEM led to increasing incorporation of
the reagent: NBD1-581 which contains a single cysteine residue,
at position 430 inside the Walker motif A, maximally incorporated 1 mol
of NEM/mol when using a NEM concentration in the range 0.8-2
mM. The inset to Fig. 6indicates that NEM
altered MANT-ATP interaction by increasing its K
value up to 6-fold.
Figure 5: MANT-ATP binding to NBD1-581 by extrinsic or intrinsic changes in fluorescence. A, spectral modification of MANT-ATP extrinsic fluorescence. The fluorescence of 5 µM MANT-ATP was measured after excitation at 350 nm in 1 ml of dialysis buffer in the absence of protein (middle curve) or the presence of 4.6 µM purified NBD1-581 (upper curve), and the differential spectrum corresponding to bound TNP-ATP was determined (lower curve); arrows indicate the different wavelength values corresponding to maximal emission, 432 nm in the case of bound MANT-ATP. B, concentration-dependent binding of MANT-ATP by monitoring the increase in fluorescence at 432 nm. C, intrinsic fluorescence of NBD1-581 and quenching upon MANT-ATP binding. The excitation spectrum of 1 µM NBD1-581 (left side) was recorded by setting emission at 300 nm, and the emission spectrum (right side) was recorded upon excitation at 275 nm either in the absence of nucleotide (solid line) or after addition of 70 µM MANT-ATP (dashed line).
Figure 6:
Alteration of MANT-ATP binding by chemical
modification with NEM. NBD1-581 (10 µM) was
incubated for 60 min at 25 °C in the absence of NEM (), and 10
mM dithiothreitol was added. The mixture was diluted 10-fold
with dialysis buffer into the spectrofluorometric cuvette, and the
concentration-dependent binding of MANT-ATP, from 5 to 80
µM, was monitored by quenching of the intrinsic emission
fluorescence as illustrated in Fig. 5C. Inset,
NBD1-581 was incubated with increasing concentrations of NEM from
0.1 to 2 mM (
) and assayed for MANT-ATP binding by
quenching of intrinsic fluorescence; the corresponding K
values were determined and plotted as a
function of covalently bound [
C]NEM measured as
described under ``Experimental
Procedures.''
Fig. 7shows that incubation of the
domain with a low 8-azido-[-
P]ATP
concentration in the presence of magnesium ions followed by ultraviolet
irradiation led to covalent incorporation of the ATP analogue, as
visualized by autoradiography (lane 1). The labeling was
partially prevented by either 50 µM TNP-ATP (lane
2) or 8 mM ATP (lane 3), confirming that all
nucleotides bind to the same site. NEM modification very efficiently
prevented photoaffinity labeling since the spot of autoradiography was
no longer visible (lane 4 as compared to lane 5).
Figure 7:
Photoaffinity labeling with 8-azido-ATP.
NBD1-581 was preincubated or not with nucleotides or NEM and
photolabeled with 8.7 µM 8-azido-[-
P]ATP as described under
``Experimental Procedures.'' After SDS-polyacrylamide gel
electrophoresis, proteins were stained with Coomassie Blue (A)
and then submitted to autoradiography (B). Lane 1,
control without preincubation; lane 2, preincubation with 50
µM TNP-ATP; lane 3, preincubation with 8 mM ATP; lane 4, pretreatment for 1 h with 1 mM NEM
followed by 10 mM dithiothreitol; lane 5, control
without pretreatment supplemented with 10 mM dithiothreitol; MW, molecular mass markers.
When assayed at a high protein concentration in the presence of
millimolar MgATP, NBD1-581 exhibited a low but significant ATPase
activity (Fig. 8A); linear double-reciprocal plots
allowed graphical estimation of a V value of 25
nmol of ATP hydrolyzed/min
mg of protein, corresponding to a
turnover number of 0.5 min
, and a K
value for MgATP of 2.1 mM (B, empty circles).
Addition to the assay medium of TNP-ATP at 5 µM (empty
squares) or 10 µM(closed squares) produced
a competitive inhibition of ATP hydrolysis. Chemical modification by
NEM inhibited ATPase activity (A, closed circles): the
inhibition was more pronounced at low substrate concentration (74% at
0.12 mM MgATP) as compared to high concentration (45% at 4.8
mM MgATP), and double-reciprocal plots indicated that the K
MgATP was increased about 5-fold (not shown
here).
Figure 8:
Kinetics of ATPase activity. A,
the NBD1-581 domain (100 µg of protein) was preincubated
() or not (
) with 2 mM NEM for 60 min, supplemented
with 10 mM dithiothreitol, and assayed at 30 °C with
increasing concentrations of MgATP (see ``Experimental
Procedures''). B, double reciprocal plots of the domain
activity in the absence of TNP-ATP (
) or in the presence of a 5
µM (
) or 10 µM (
)
concentration.
The original aspects of this paper concern the first preparation of a soluble N-terminal nucleotide-binding domain from P-glycoprotein, the measurement of its interaction parameters with substrate ATP and analogues, and the characterization of the cysteine residue located inside the Walker motif A.
Previous evidences for
nucleotide interaction were more indirect, based on site-directed
mutagenesis altering overall ATP hydrolysis and related drug
transport(11, 51) , on the ATPase activity of
membrane-bound N-terminal half (12, 15) and on
chemical modifications of whole P-glycoprotein(50) . Using the
NBD1-581 domain, we find here an apparent K value for ATP in the absence of magnesium ions, to avoid
hydrolysis, about 3-fold higher than the K
MgATP
for ATPase activity. The latter value, 2.1 mM, is not very
different from those obtained with membrane-bound or purified whole
P-glycoprotein(14, 15, 16, 17, 52, 53) but
contrasts with the unexpectedly high value, 20 mM, reported
for NBD2 fused to the maltose-binding protein(20) . The ability
of purified NBD1 to hydrolyze ATP appears to be an intrinsic activity
of the domain since: (i) the maximal rate, 25 nmol of ATP hydolyzed/min
mg of protein, is comparable to that reported for recently
characterized NBD1 from CFTR (45) and for NBD2 fused to the
maltose-binding protein(20) ; (ii) the activity is
competitively inhibited by TNP-ATP, at micromolar concentrations
related to the analogue binding as determined by fluorescence; (iii) it
is inhibited by NEM, as whole P-glycoprotein (13, 16, 17, 53) and its N-terminal
half(15) , which increases the K
MgATP
similarly as it increases the K
MANT-ATP. The
maximal rate is, however, much lower as compared to domains from the
bacterial ABC transporters HlyB (21) and MalK(54) . It
is also much lower than whole purified P-glycoprotein (17, 55, 56) which suggests that full
activity would require critical interactions with NBD2 and/or
transmembrane domains, and possibly with the phosphorylatable linker
region as proposed for the CFTR regulatory domain(57) . The
fact that the same activity was obtained upon renaturation of
NBD1-581 inclusion bodies favors that the domain overproduced and
purified as a soluble protein retained a fully native conformation.
In Chinese hamster membrane P-glycoprotein, two cysteine residues, among a total amount of seven, were derivatized by radioactive NEM when complete inhibition of the drug-stimulated ATPase activity was achieved, the radioactivity being equally distributed in each half of the molecule(50) . The results reported here with purified mouse NBD1 strongly support that Cys-430 inside the Walker motif A is the essential residue derivatized by NEM in the N-terminal half of whole P-glycoprotein. This finding corroborates the very recent report showing that reintroduction of a single cysteine residue in the Walker motif A of the N-terminal half of a Cys-less P-glycoprotein mutant restores the sensitivity of the drug-stimulated ATPase to NEM inhibition(58) . Accordingly, the ATPase activity of purified HlyB nucleotide-binding domain, which does not contain any cysteine in its Walker motif A, is not inhibited by NEM(21) . In addition, we show here that the inhibition of the domain-intrinsic ATPase activity by NEM, in the absence of drugs, is due to alteration of substrate ATP binding.
Work is in progress concerning NBD1 structural and biochemical properties and its in vitro interactions with other recombinant domains of P-glycoprotein.