Báo cáo khoa học: Functional symmetry in the isolated domain demonstrated by N-ethylmaleimide labelling pdf

The nucleotide-binding domains of P-glycoprotein
Functional symmetry in the isolated domain demonstrated by
N
-ethylmaleimide
labelling
Georgina Berridge
1
, Jennifer A. Walker
1
, Richard Callaghan
1
and Ian D. Kerr
1,2
1
Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, UK;
2
School of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham, UK
The two nucleotide-binding domains (NBDs) of a number of
ATP-binding cassette (ABC) transporters have been shown
to be functionally dissimilar, playing different roles in the
transport process. A high degree of co-operativity has been
determined for the NBDs of the human multidrug trans-
porter, P-glycoprotein. However, the issue of functional
symmetry in P-glycoprotein remains contentious. To
address this, the NBDs of P-glycoprotein were expressed and
purified to 95% homogeneity, as fusions to maltose-binding
protein. The NBDs were engineered to contain a single
cysteine residue in the Walker-A homology motif. Reactivity
of this cysteine residue was demonstrated by specific, time-
dependent, covalent labelling with N-ethylmaleimide. No
differences in the rates of labelling of the two NBDs were

observed. The relative affinity of binding to each NBD was
determined for a number of nucleotides by measuring their
ability to effect a reduction in N-ethylmaleimide labelling. In
general, nucleotides bound identically to the two NBDs,
suggesting that there is little asymmetry in the initial step of
the transport cycle, namely the recognition and binding of
nucleotide. Any observed functional asymmetry in the intact
transporter presumably reflects different rates of hydrolysis
at the two NBDs or interdomain communications.
Keywords: ABC transporter; cysteine; functional symmetry;
maleimide; Walker-A.
ATP-binding cassette (ABC) transporters are multidomain
membrane proteins, responsible for the controlled efflux
and influx of substances (allocrites) across cellular mem-
branes [1]. They are minimally composed of four domains,
with two transmembrane domains (TMDs) responsible for
allocrite binding and transport and two nucleotide-binding
domains (NBDs) responsible for coupling the energy of
ATP hydrolysis to conformational changes in the TMDs
[2,3]. A detailed understanding of ABC transporter-medi-
ated allocrite flux requires the delineation of the interactions
between the four domains.
Studies aimed at elucidating aspects of the transport cycle
of P-glycoprotein have demonstrated that both NBDs are
capable of ATP hydrolysis [4], that inhibition of hydrolysis
at one NBD effectively abrogates hydrolysis at the other [5],
andthathydrolysisatthetwoNBDsmayoccurinan
alternative fashion [6]. However, whether the two NBDs
have a functionally identical role in the transport cycle, or if
they are functionally nonequivalent remains a contentious

issue. The reaction pathway proposed for P-glycoprotein
involves ATP binding, hydrolysis, release of phosphate, and
release of ADP. As both ATP hydrolysis and phosphate
release appear to be rapid events [4], the rate-limiting step in
this scheme is proposed to be either ATP association or
ADP dissociation. Asymmetry in either of these events
would be a critical component of overall functional
asymmetry. Approaches to addressing this issue in
P-glycoprotein (for example, determining the effects on
ATPase activity subsequent to mutagenesis of equivalent
residues in the N-terminal and C-terminal NBD) provide
data both in favour [7–10] and against [11–13] functional
asymmetry. The reasons for the discrepancies are unclear.
This is in contrast with many other ABC transporters, for
which there is evidence that the two NBDs, although highly
similar in sequence, may adopt different functional roles in
the transport cycle. Pertinent data have been presented for
the multidrug resistance-associated protein (MRP1 [14,15]),
the transporter associated with antigen processing (TAP
[16,17]), the yeast a-factor transporter (Ste6 [18]), and the
cystic fibrosis transmembrane conductance regulator
[19,20].
The purpose of this study is to examine the NBDs of
human P-glycoprotein in an in vitro system to determine if
asymmetry of NBDs can be detected in studies of the
isolated domain. We investigate asymmetry at the initial
step in the catalytic cycle, namely nucleotide binding.
Numerous investigations have demonstrated that nucleotide
binding is a key event in the transport cycle. For example,
changes in immunoreactivity [21], drug affinity [22], pro-

tease sensitivity [23], and tertiary structure [10,24] are all
associated with nucleotide binding. Furthermore, binding of
nucleotide and the associated conformational change have
Correspondence to I. Kerr, School of Biomedical Sciences,
University of Nottingham, Queen’s Medical Centre,
Nottingham NG7 2UH, UK.
Fax: + 44 115 970 9969, Tel.: + 44 115 875 4682,
E-mail:
Abbreviations: ABC, ATP-binding cassette; p[NH]ppA, adenosine
5¢-[b,c-imido]-triphosphate; MBP, maltose-binding protein;
MIANS, 2-(4¢-maleimidylanilino)naphthalene-6-sulfonic acid;
NBD, nucleotide-binding domain; TAP, transporter associated
with antigen processing; TMD, transmembrane domain.
(Received 30 October 2002, revised 20 January 2003,
accepted 10 February 2003)
Eur. J. Biochem. 270, 1483–1492 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03514.x
been demonstrated to occur within isolated NBDs, suggest-
ing that such domains are a useful model system for
analysing nucleotide binding in the intact transporter.
Structural investigations of bacterial NBDs have revealed
conformational differences in nucleotide-free and nucleo-
tide-bound forms [25,26], which can be mirrored by
molecular modelling studies (J. D. Campbell, I. D. Kerr &
M. S. P. Sansom unpublished results). These studies are in
agreement with the hypothesis that nucleotide can bind to a
single NBD, as a precursor to NBD dimerization [27,28].
In this study we utilized the ability of thiol-reactive
reagents to label endogenous cysteines located within the
ATP-binding pocket of the NBDs. Previous data have
demonstrated that the Walker-A cysteine residues are both

accessible to thiol-specific probes, and that derivitization of
these residues prevents ATP hydrolysis [5,8,29]. Recent
structural determinations of related ABC transporter NBDs
(for example, HisP [30]) confirm that the equivalent residue
exposes its side chain to the nucleotide-binding pocket [3].
We describe the engineering, expression and purification of
single-cysteine-containing proteins comprising either the
C-terminal NBD or the N-terminal NBD as fusions to
Escherichia coli maltose-binding protein (MBP). Having
demonstrated accessibility of the unique Walker-A cysteine
residue in these fusion proteins, we investigated if nucleotide
preincubation could prevent derivitization by maleimide
reagents. Our results show that the N-terminal and
C-terminal NBDs are functionally similar in their ability
to bind ATP and ADP, but show some differences in their
ability to bind other nucleotides, including adenosine
5¢-[b,c-imido]-triphosphate (p[NH]ppA). These differences
may be attributable to the small but significant sequence
differences between the N-terminal and C-terminal NBDs, a
hypothesis that we have investigated further by molecular
modelling. The relevance of our data to the understanding
of P-glycoprotein functional asymmetry is discussed.
Experimental Procedures
Reagents and chemicals
N-Ethylmaleimide was obtained from Sigma (Poole,
Dorset, UK). N-[
3
H]Ethylmaleimide (specific activity
40–60 CiÆmmol
)1

) was from NEN Biochemicals (Zaventem,
Belgium). Restriction enzymes were from New England
Biolabs (Hitchin, Herts, UK), and PCR primers were from
Sigma-Genosys (Cambridge, UK). The following nucleo-
tides (Sigma unless stated) were used in N-[
3
H]ethylmale-
imide labelling and ATPase assays: disodium ATP,
disodium 2-deoxy-ATP, sodium ADP, tetralithium
p[NH]ppA (Calbiochem, Nottingham, UK), disodium
CTP, lithium GTP, sodium dTTP, trisodium ITP. 2-(4¢-
Maleimidylanilino)naphthalene-6-sulfonic acid (MIANS)
was obtained from Molecular Probes (Eugene, OR,
USA). All other reagents were of analytical grade or better.
Generation of fusion proteins
NBDs of human P-glycoprotein were fused to the
C-terminus of E. coli MBP in the vector pMal-C2x (New
England Biolabs). The N-terminal NBD was amplified by
PCR using DNA encoding wild-type human P-glycoprotein
as template. The forward and reverse oligonucleotide
sequences are 5¢-GAAGAGTGGGCAAC
GGATCCGAT
AATATTTAAG and 5¢-CATTTCCTGCTGT
CTGCAG
TCAGACAAGTTTGAAG, respectively. The restriction
sites encoded within these primers are underlined. The
C-terminal NBD was amplified from DNA encoding
cysteine-less P-glycoprotein [31] into which Cys1074 (in
the Walker-A motif) had been re-introduced by site-directed
mutagenesis (Altered Sites, Promega). The mutagenic

primer had the sequence 5¢-GGCAGCAGTGGC
TGTGG
GAAGAGCACAG, in which the introduced cysteine
codon is underlined. After introduction of the Cys1074,
the C-terminal NBD was amplified using forward and
reverse primers 5¢-CAGCACGGAAGGC
GAATTCCCG
AACACATTG and 5¢-CTTTGTTCCAGC
CTGCAGT
CAGACCATTGAAAA, respectively (restriction cloning
sites underlined). N-terminal and C-terminal NBDs were
cloned into the pMal-C2X vector at the BamHI/PstIand
EcoRI/PstI sites, respectively, to generate plasmids pMal-
C2x-Nter and pMal-C2x-Cter. Sequences of the NBDs were
verified by DNA sequencing (Biochemistry Department,
University of Oxford), which also confirmed the fidelity of
the mutagenesis reaction.
Protein expression
pMal-C2x-Nter and pMal-C2x-Cter were transformed into
chemically competent E. coli BL21.kDE3 [32]. Single colon-
ies were inoculated into 5 mL Luria–Bertani broth supple-
mented with 100 lgÆmL
)1
ampicillin and grown overnight at
37 °C with shaking at 200 r.p.m., and then diluted 1 : 80 into
400 mL Luria–Bertani broth/ampicillin. Growth was con-
tinued at 37 °C until an A
600
of 0.5 was achieved. Cultures
were then cooled to 25 °C and induced with 0.2 m

M
isopropyl thio-b-
D
-galactoside. After 3–4 h shaking at
25 °C, bacteria were harvested by centrifugation at 3000 g.
Purification of fusion proteins
Bacterial cell pellets were resuspended in 10 mL lysis buffer
(50 m
M
Tris/HCl, 150 m
M
NaCl, 20% glycerol, pH 7.4) by
vortex-mixing. Bacteria were lysed by sonication (10 · 10 s
bursts on ice). Lysis was verified by examination under a
microscope. Bacterial lysates were cleared by a low-speed
centrifugation (10 000 g for 10 min). Soluble proteins were
isolated from this supernatant by centrifugation at 60 000 g
for 60 min at 4 °C (Beckman TLA 100 rotor). Soluble
protein was diluted to a concentration of 3 mgÆmL
)1
and
incubated with pre-equilibrated amylose resin (New Eng-
land Biolabs) at a protein/resin ratio of 4 : 1 (w/v) for
30 min at room temperature. Resin and protein were loaded
on to BioSpin columns (Bio-Rad), and unbound material
was discarded. Nonspecifically bound proteins were subse-
quently removed by four washes with 2 mL lysis buffer
supplemented with 10 l
M
maltose. Bound fusion proteins

were eluted by subsequent washes of 2 mL lysis buffer
containing 1 m
M
maltose. Aliquots of all fractions were
analysed by SDS/PAGE, and those containing fusion
proteins were pooled and concentrated under nitrogen with
an Amicon stirred-cell using a PM10 membrane (10-kDa
cut-off). ATPase activities of purified fusion proteins were
determined by colorimetric assay [33].
1484 G. Berridge et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Labelling with
N
-ethylmaleimide
N-[
3
H]Ethylmaleimide (specific radioactivity 40–60 CiÆ
mmol
)1
,1mCiÆmL
)1
) is supplied in pentane to which was
added 250 lL dimethyl sulfoxide (1 : 4 the original vol-
ume), and the pentane was evaporated under a stream of
nitrogen. N-[
3
H]Ethylmaleimide (4 mCiÆmL
)1
)wasthen
frozen in aliquots at )20 °C. N-[
3

H]Ethylmaleimide treated
in this way was stable without significant loss in the intensity
of protein labelling for 4–6 weeks (data not shown). On
longer-term storage, we found that greater exposure times
were necessary to achieve the same labelling intensity,
suggesting that exchange of the tritium label may occur with
the solvent during storage over 2 months.
The time dependence of labelling was measured by
incubating protein (1.5 lg) with N-[
3
H]ethylmaleimide
(0.6 l
M
, determined by liquid-scintillation counting) for
increasing times at 22 °C. The reaction was stopped by the
addition of protein loading sample buffer containing a
minimum 10-fold molar excess of a-mercaptoethanol.
Control experiments established that this excess prevented
further labelling. Proteins were resolved by SDS/PAGE
(10% gels), fixed (propan-2-ol/acetic acid/water,
25 : 10 : 65, v/v/v), and soaked in AMPLIFYÒ (Amer-
sham, UK). Gels were then dried on to Whatman 3 mm
paper and exposed to photographic film (Kodak, MR1
film) at )80 °C.
To investigate if nucleotide preincubation can prevent
N-[
3
H]ethylmaleimide labelling, 1.5 lg protein in lysis
buffer, supplemented with 5 m
M

MgCl
2
, was incubated
with increasing concentrations of nucleotide at 37 °Cfor
30 min. N-[
3
H]Ethylmaleimide was then added to a final
concentration of 0.6 l
M
and incubated at 22 °C for 15 min.
Samples were then subjected to SDS/PAGE and autoradio-
graphy as described above. The final concentration of
dimethyl sulfoxide in any experiment never exceeded 2.5%
(v/v). Parallel controls demonstrated that the addition of
dimethyl sulfoxide to 25% (v/v) did not affect N-[
3
H]ethyl-
maleimide labelling.
Autoradiographs were analysed using freely available
densitometry software (Scion Image, www.scioncorp.com).
All images were scanned at a resolution of 300 dpi and
analysed without further modification. Exposures of the
same gel for different time periods were employed to
ensure that saturation of the densitometric signal had not
occurred. For the time course of N-[
3
H]ethylmaleimide
labelling experiments, the most intensely labelled band
was used as a 100% reference. Plots of time against
percentage intensity were obtained, and the single-phase

exponential association curve was fitted by nonlinear
regression using
PRISM
(GraphPad, San Diego, CA, USA)
to the equation:
y ¼ y
max
Àð1 À exp
Àkt
Þ
where y ¼ percentage saturation, t ¼ time, k ¼ time con-
stant for labelling (T
0.5
¼ 0.69/k).
For investigations on nucleotide protection of N-[
3
H]eth-
ylmaleimide labelling, the most intensely labelled band was
designated the reference signal. All other data points were
quantified as the percentage intensity of the 100% reference
signal. Plots of nucleotide concentration against percentage
intensity were obtained and the sigmoidal dose–response
curve was fitted by nonlinear regression, using Prism, to the
equation:
y ¼ bottom þ
ðtop À bottomÞ
1 þ 10
ðlog IC
50Àlog x
Þ

n
where y ¼ percentage of reference signal, x ¼ nucleotide
concentration, bottom ¼ minimum labelling inten-
sity, top ¼ saturation of labelling, IC
50
¼ concentration
required to reduce labelling intensity to 50% of its
maximum, and n ¼ Hill slope.
Data were fitted with either a Hill slope of 1.0 or a
variable Hill slope. An F test was used to determine if the
data were best fit by an equation containing a fixed or
variable slope. Statistical comparison of T
0.5
and IC
50
values was performed using an unpaired Student’s t test. In
all cases a value of P < 0.05 was considered significant.
Unless indicated otherwise, all data are presented as the
mean ± SEM.
Molecular modelling of P-glycoprotein NBDs
Sequences of P-glycoprotein NBDs and TAP1 were aligned
using
CLUSTALW
[34]. A series of 10 homology models of
each NBD was constructed, using the program
MODEL-
LER
_6
V
2 [35], and employing the crystal structure of TAP1

as a structural template [36]. TAP1 is an appropriate choice
of template as it shares a high degree of sequence identity to
the NBDs of P-glycoprotein, and is a member of the same
subfamily of ABC transporters (ABCB). However, the
reported crystal structure of TAP1 does not contain ATP.
Co-ordinates for ATP were added to TAP1 by least-squares
superimposition of TAP1 and HisP, which does contain a
bound ATP molecule [30]. Analysis of the individual
P-glycoprotein NBD models in terms of stereochemistry
and local packing enabled the selection of a preferred
N-terminal and C-terminal NBD model. Structural analysis
was performed using
WHAT
-
CHECK
[37] and structural
diagrams were produced using
MOLSCRIPT
[38].
Results
Expression and purification of fusion proteins
Previously, we and others have shown that expression
in E. coli of the N-terminal and C-terminal NBDs of
P-glycoprotein as soluble proteins is very difficult to achieve
[39–40, I. D. Kerr, G. Berridge & R. Callaghan, unpub-
lished results]. To circumvent this, we expressed NBDs as
fusions to the C-terminus of E. coli MBP. Subsequent
functional assays utilized covalent attachment of probes to
cysteine residues in the NBDs. E. coli MBP is devoid
of cysteine residues [42], and the N-terminal NBD of

P-glycoprotein contains only a single cysteine in the
Walker-A motif [43]. Thus, MBP-NBD-Nter is a 75-kDa
fusion protein containing a single cysteine residue located in
the ATP-binding pocket. The production of a similar fusion
protein containing the C-terminal NDB of P-glycoprotein is
hampered by the fact that the C-terminus of P-glycoprotein
contains three endogenous cysteine residues. To circumvent
this we employed, as a PCR template, DNA encoding
cysteine-less P-glycoprotein [31] in which the native cysteine
at position 1074 was re-introduced by site-directed
Ó FEBS 2003 Functional symmetry of P-glycoprotein NBDs (Eur. J. Biochem. 270) 1485
mutagenesis. Thus, both MBP-NBD-Nter and MBP-NBD-
Cter are single-cysteine-containing proteins, in which the
cysteine residue is in the Walker-A motif.
MBP-NBDs are expressed to high level in a soluble form
(Fig. 1, lane 1), % 60% of which binds to the amylose resin.
Purification of the fusion proteins is facilely achieved with
affinity chromatography (Fig. 1). This one-step purification
produced a purity of % 95%. Typically, a 400-mL bacterial
culture yielded 1–2 mg fusion protein at a final concentration
of 0.5–1.0 mgÆmL
)1
. This contrasts with our attempts to
purify the N-terminal NBD in isolation as a soluble protein
with a hexa-histidine tag, which requires a two-step purifi-
cation to remove contaminating proteins (I. D. Kerr,
G. Berridge & G. Callaghan, unpublished results). The
oligomerization state of the fusion protein was investigated
chromatographically. MBP-NBD-Cter or MBP-NBD-Nter
was incubated with amylose resin for 30 min at room

temperature. A purified, N-terminal NBD (free of MBP; I. D.
Kerr, G. Berridge & G. Callaghan, unpublished results) was
then added to the immobilized fusion protein. The N-ter-
minal NBD passed through the column, and was present
entirely in the flow-through fraction, demonstrating that
under the conditions used there was no interaction with
MBP-NBDs (data not shown). The chromatographic buffer
conditions were identical with those used during N-ethylma-
leimide labelling studies, suggesting that the fusion proteins
are monomeric in solution, under these mild ionic conditions.
Although a Factor Xa cleavage site is present in the linker
between the two halves of the fusion protein, attempts to
cleave NBD from MBP consistently displayed concomitant
degradation of the NBD (there are two potential Factor
Xa (Gly-Arg) sites one in the N-terminal and one in the
C-terminal NBD). Similarly, the pMal-C2 plasmid is
available with a subtilisin cleavage site in the linker region.
NBDs were cloned into this plasmid, and fusion proteins
were expressed. However, attempts at enzymatic hydrolysis
of the resultant protein were ineffective, presumably because
of inaccessibility of the cleavage site.
Examination of the ATP-binding pockets of bacterial
NBDs for which structural data are available (e.g. HisP,
Rad50, MalK [30,44,45]) demonstrates that the side chain of
the equivalent residue (Ser43 in HisP, Ser34 in Rad50, and
Cys40 in MalK) is located within the ATP-binding pocket.
Indeed, the mean distance between the equivalent side chain
and the a-phosphate of bound nucleotide (or pyrophos-
phate in the case of MalK) is only 5.2 A
˚

. This suggests that
(a) the single cysteine in MBP-NBD proteins may be
susceptible to derivatization, and (b) occupancy of the
nucleotide-binding site could alter the accessibility and
reactivity of this cysteine. Therefore, the purified single-
cysteine-containing fusion proteins were examined for their
ability to bind N-ethylmaleimide.
The single cysteine residue is accessible
to
N
-ethylmaleimide
The initial experiments provided a detailed characterization
of the binding of N-[
3
H]ethylmaleimide to MBP-NBD
fusion proteins. Time, temperature and concentration
dependence of N-[
3
H]ethylmaleimide labelling was investi-
gated to optimize conditions for nucleotide preincubation
experiments described below. It was demonstrated that
effective labelling of fusion proteins could be obtained at
pH 7.4, 22 °C and approximately equimolar N-ethylmale-
imide and protein. Use of these assay conditions avoids
nonspecific labelling of noncysteine residues (e.g. histidine)
as previously described [46]. In support of this, N-[
3
H]ethyl-
maleimide was shown not to label MBP on its own, or
fusion proteins derived from cysteine-less P-glycoprotein

(data not shown), consistent with the absence of cysteine
residues from these.
The representative binding data presented in Fig. 2 for
N-[
3
H]ethylmaleimide investigates the time course of deri-
vatization of the cysteine residue in the ATP-binding pocket
and demonstrates its accessibility to N-[
3
H]ethylmaleimide.
The data were best fit to a single-phase exponential (again
consistent with N-ethylmaleimide reacting with a single thiol
residue), analysis of which produced a half-time (T
0.5
)for
the association of N-[
3
H]ethylmaleimide with MBP-NBD-
Nter of 30.6 ± 1.5 min (n ¼ 4), and with MBP-NBD-Cter
of 38.0 ± 4.3 min (n ¼ 4). Unpaired t test demonstrates no
difference in the T
0.5
for N-[
3
H]ethylmaleimide labelling of
the two fusion proteins under these conditions.
Preincubation with ATP but not ADP prevents
N
-[
3

H]ethylmaleimide labelling
The data above show that the cysteine in MBP-NBDs is
accessible to modification by N-ethylmaleimide, confirming
our initial hypothesis. Our second hypothesis, that nucleo-
tide occupancy of the ATP-binding pocket would be
manifested as a reduction in N-[
3
H]ethylmaleimide labelling,
was investigated in subsequent experiments. As N-ethyl-
maleimide binding is irreversible, whereas nucleotide binding
is reversible, we used constant experimental conditions to
enable comparison of the effect of different nucleotides on
N-[
3
H]ethylmaleimide labelling of the two fusion proteins.
To investigate potential asymmetry in the two NBDs, we
preincubated fusion proteins with various concentrations of
nucleotides, allowing them to come to equilibrium before
Fig. 1. Purification of MBP-NBD fusion protein. MBP-NBD-Nter was
purified from E. coli BL21 kDE-3 as described in Experimental pro-
cedures. Lanes S (soluble proteins) and F (flow-through) contain 5 lg
protein, whereas all other lanes contain 100 lL (1/20th of each frac-
tion) precipitated by trichloroacetic acid. W1–W4 indicate four washes
with 10 l
M
maltose. E1–E6 represent elution fractions in 1 m
M
maltose. Samples were resolved by SDS/PAGE (10% gel), and stained
with Coomassie blue. The approximate positions of molecular-mass
markers are indicated on the right.

1486 G. Berridge et al.(Eur. J. Biochem. 270) Ó FEBS 2003
adding 0.6 l
M
N-[
3
H]ethylmaleimide. This reaction was
allowed to proceed for 15 min at 22 °C, over which time any
inhibition of N-[
3
H]ethylmaleimide labelling, caused by the
presence of nucleotide in the binding site, would be evident.
Characterization of MBP-NBDs by the sensitive Chifflet
assay [33] demonstrated that there was no NTPase activity of
the fusion proteins (data not shown). Thus, any effects on
N-[
3
H]ethylmaleimide labelling are due to nucleotide bind-
ing only. Nucleotide concentrations of up to 3.5 m
M
were
employed. Higher concentrations of nucleotides caused a
significant alteration of the pH, which may affect the
specificity of maleimide reactivity [46]. Example results of
experiments conducted with ATP are shown in Fig. 3A,B,
and similar results were obtained in multiple independent
experiments with different batches of fusion proteins. The
data, fitted by a sigmoid dose–response equation, are plotted
as a function of ATP concentration in Fig. 3C. ATP at
concentrations higher than 3 m
M

was able to completely
prevent N-ethylmaleimide labelling under the reaction
conditions used. The mean data, obtained in at least eight
experiments, return IC
50
values for the inhibition of
N-[
3
H]ethylmaleimide labelling for MBP-NBD-Nter of
1.8 ± 0.2 m
M
(n ¼ 8) and for MBP-NBD-Cter of 2.3 ±
0.2 m
M
(n ¼ 9). The difference in the potency of reduction
in N-[
3
H]ethylmaleimide labelling between the N-terminal
and C-terminal NBDs was not significant. Pretreatment with
2-deoxy-ATP, an effective substitute in ATPase reactions, is
also able to confer protection against N-[
3
H]ethylmaleimide
labelling with indistinguishable values of potency and extent
of labelling diminution to ATP (Table 1).
In contrast, ADP did not offer any protection against
N-[
3
H]ethylmaleimide labelling (Table 1), with negligible
displacement at concentrations up to 3.5 m

M
in either the
N-terminal or C-terminal NBD. This result was independ-
ently confirmed by fluorescence experiments in which
MIANS was used as the thiol-reactive agent. Again, no
reduction in MIANS labelling of MBP-NBDs was demon-
stratedatconcentrationofupto3.5m
M
ADP (data not
shown).
Preincubation with other nucleotides reveals subtle
functional differences between N-terminal
and C-terminal NBDs
Data in the previous section demonstrated that N-[
3
H]eth-
ylmaleimide labelling of MBP-NBD fusion proteins was
Fig. 3. ATP protects against N-[
3
H]ethylmaleimide labelling of fusion
proteins. Incubation of proteins with nucleotide and subsequent
labelling with N-[
3
H]ethylmaleimide was carried out as described in the
text. (A) MBP-NBD-Nter and (B) MBP-NBD-Cter preincubated with
increasing concentrations (0–3.5 m
M
) of ATP (displayed beneath each
lane). The approximate position of the 80-kDa molecular-mass marker
is denoted by a solid line. The sigmoidal dose–response curve fits to the

data are shown in (C). (j) MBP-NBD-Nter; (h) MBP-NBD-Cter.
Fig. 2. Time-dependence of labelling of fusion proteins by N-[
3
H]ethyl-
maleimide. Labelling of MBP-NBD was carried out as described in the
text. (A) MBP-NBD-Nter and (B) MBP-NBC-Cter labelling over time
(in minutes displayed beneath each lane). The approximate position of
the 80-kDa molecular-mass marker is denoted by a solid line. (C)
Percentage saturation plotted as a function of labelling time. Data
points (fitted to a single exponential equation) are derived from den-
sitometric analysis of the data in (A) and (B). (j) MBP-NBD-Nter;
(h) MBP-NBD-Cter.
Ó FEBS 2003 Functional symmetry of P-glycoprotein NBDs (Eur. J. Biochem. 270) 1487
differentially affected by the hydrolysable substrate of
P-glycoprotein (ATP) and the release product (ADP).
However, there are no apparent differences between the
N-terminal and C-terminal NBDs to bind nucleotide. To
further characterize the molecular properties of each NBD,
the potency of a number of different nucleotides to prevent
N-[
3
H]ethylmaleimide labelling was determined. The data
for the effects of nonadenine-containing nucleotides is
shown in Table 2. Of the compounds examined, the only
nucleotide able to confer full protection against the deriva-
tization of the cysteine residue in Walker-A was CTP
(Table 2). However, the respective potencies to prevent
labelling were not significantly different between the N-ter-
minal NBD (IC
50

¼ 2.2 ± 0.2 m
M
) and the C-terminal
NBD (IC
50
¼ 2.3 ± 0.3 m
M
). Neither dTTP nor ITP was
able to fully prevent N-[
3
H]ethylmaleimide labelling by
preincubation at concentrations as high as 3.5 m
M
nucleo-
tide, indicating that these nucleotides have a much reduced
affinity for the NBDs of P-glycoprotein, compared with
ATP or CTP.
The most striking differences in N-[
3
H]ethylmaleimide
labelling between the two NBDs was observed with
p[NH]ppA and GTP. GTP preincubation, at concentra-
tions up to 3.5 m
M
did not protect against N-[
3
H]ethyl-
maleimide labelling of the MBP-NBD-Nter protein. In
contrast, 44 ± 8% protection was seen for labelling of
MBP-NBD-Cter at the highest achievable concentration of

3.5 m
M
. Thus, the data obtained with GTP demonstrates
that the two NBDs contain subtle differences in their
nucleotide-binding pocket that are discriminated by the
guanine nucleotide.
Further differences were highlighted by results obtained
using p[NH]ppA protection against N-[
3
H]ethylmaleimide
labelling of the two NBDs (Fig. 4). Whereas N-[
3
H]ethyl-
maleimide labelling of MBP-NBD-Nter is completely
prevented by p[NH]ppA over the experimental nucleotide
concentration range (IC
50
¼ 0.9 ± 0.1 m
M
), labelling of
MBP-NBD-Cter is only 41 ± 4% prevented at the highest
nucleotide concentration. This suggests that the C-terminal
NBD interacts differently with p[NH]ppA from the N-ter-
minal NBD.
Table 2. Inhibition of N-[
3
H]ethylmaleimide labelling by nonadenosine nucleotides. All experimental details are identical with those given in Table 1.
Where full protection from N-[
3
H]ethylmaleimide labelling was observed, an IC

50
is presented, otherwise the mean inhibition of labelling observed
at the highest nucleotide concentration (3.5 m
M
) is given. N, number of experiments with data presented as the mean ± SEM. An asterisk denotes
that the inhibition of N-[
3
H]ethylmaleimide is significantly different between MBP-NBD-Nter and MBP-NBD-Cter (P <0.05).IC
50
parameter
not determined, as it was not possible to fit the data to a dose-response equation, denoted by n/a.
GTP ITP dTTP CTP
Nter Cter Nter Cter Nter Cter Nter Cter
IC
50
n/a n/a n/a n/a n/a n/a 2.2 ± 0.2 2.3 ± 0.3
Inhibition (%) 0 ± 10* 48 ± 3* 44 ± 8 27 ± 6 42 ± 4 41 ± 10 93 ± 7 97 ± 3
N 4 4 6454 6 7
Fig. 4. p[NH]ppA reacts differently with N-terminal and C-terminal
MBP-NBD fusion proteins. Incubation of proteins with nucleotide and
subsequent labelling with N-[
3
H]ethylmaleimide was carried out as
described in Experimental Procedures. The mean ± SEM is shown.
(j) MBP-NBD-Nter; (h) MBP-NBD-Cter.
Table 1. Inhibition of N-[
3
H]ethylmaleimide labelling by adenosine nucleotides. Nucleotide at various concentrations was preincubated with fusion
protein, followed by addition of N-[
3

H]ethylmaleimide. The percentage inhibition of N-ethylmaleimide labelling observed was quantified as
described in the text. Nter represents MBP-NBD-Nter; Cter represents MBP-NBD-Cter. Where full protection from N-[
3
H]ethylmaleimide
labelling was observed, an IC
50
is presented. Otherwise the mean protection from labelling observed at the highest nucleotide concentration
(3.5 m
M
) is given. N, number of experiments. Data presented as the mean ± SEM. An asterisk denotes that the inhibition of N-[
3
H]ethylmaleimide
is significantly different between MBP-NBD-Nter and MBP-NBD-Cter (P < 0.05). IC
50
parameter not determined, as it was not possible to fit the
data to a dose-response equation, denoted by n/a.
ATP dATP p[NH]ppA ADP
Nter Cter Nter Cter Nter Cter Nter Cter
IC
50
1.8 ± 0.2 2.3 ± 0.2 1.6 ± 0.2 2.0 ± 0.2 0.9 ± 0.1 n/a n/a n/a
Inhibition (%) 94 ± 4 94 + 2 89 ± 9 98 ± 2 100 ± 1* 41 ± 4* 0 ± 12 0 ± 10
N 8956 6499
1488 G. Berridge et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Discussion
In this study, we have investigated the similarity between the
two NBDs of P-glycoprotein in nucleotide binding. Differ-
ences in this initial step of the catalytic cycle would be
manifested in asymmetric roles in transport. In this work,
we used a fusion protein consisting of either the N-terminal

or C-terminal NBD of P-glycoprotein, fused to E. coli
MBP. This is necessary because of the inherent insolubility
of isolated NBDs of human ABC transporters (I. D. Kerr,
G. Berridge & R. Callaghan, unpublished results). A similar
fusion protein approach has previously been used to express
the NBDs of P-glycoprotein [47,48]. Consistent with this, we
were unable to specifically cleave NBD1 from MBP-NBD
fusions by digestion with Factor Xa. In contrast with our
data, nucleotide hydrolysis was demonstrated in the latter
study [48], although at a lower specific activity than
observed for full-length P-glycoprotein, potentially because
of the quantities of fusion protein employed in ATPase
assays. In the light of the inability of our fusion proteins to
hydrolyse nucleotide, a novel approach was used to
characterize the interaction of nucleotides with NBDs,
employing the unique, reactive cysteine residue in the
Walker-A sequence.
The two NBDs of P-glycoprotein are functionally similar
The similar time course of binding of N-[
3
H]ethylmaleimide
to the Walker-A cysteine residue in the N-terminal and
C-terminal NBDs suggests that local steric effects and
accessibility of the cysteine are identical in the two halves of
P-glycoprotein. We therefore used nucleotide protection of
N-[
3
H]ethylmaleimide labelling to investigate the inter-
actions of diverse nucleotides with P-glycoprotein NBDs.
Protection against derivatization is afforded when nucleo-

tide is in the binding pocket, and therefore the potency of
protection is a measure of binding affinity. Of course, as
nucleotide binding is a reversible process and N-ethylmale-
imide labelling is irreversible, we are unable to determine
absolute binding affinities of nucleotides from such data.
However, under constant experimental conditions, we are
able to compare the relative affinities of nucleotides. As an
alternative approach we investigated the possibility of using
8-azido-[
32
P]ATP labelling to determine relative affinities of
nucleotides. However, the maximum concentration of
commercially available 8-azido-[
32
P]ATP is only 100 l
M
in
methanol. Avoiding excess solvent in labelling experiments
would impose an upper limit on the achievable concentra-
tion of 8-azido-[
32
P]ATP of % 5 l
M
. This is two orders of
magnitude below the K
m
for 8-azido-ATP [29], suggesting
that only 2–4% of fusion protein would be labelled, thus
preventing meaningful competition binding studies.
The data we present show that the hydrolysable substrate

(ATP) and the hydrolysis product (ADP) are nonequivalent
in this system. Whereas ATP can fully inhibit maleimide
labelling (within the 15 min time course of the experiment),
ADP is incapable of preventing cysteine derivatization over
the same time period. This suggests that either ADP has a
lower binding affinity or a more rapid dissociation from the
NBD than ATP. Rapid dissociation of hydrolysis product
may be expected as part of the kinetics of the transport cycle
of the intact transporter. Furthermore, the data demon-
strate that the two NBDs interact in a similar manner with
2-deoxy-ATP and CTP. Lastly, the data provide evidence
that ITP and dTTP bind weakly, and that their respective
interaction with the N-terminal and C-terminal NBDs is
identical.
Our results are comparable to data obtained on the
inhibition of ATPase activity of full-length P-glycoprotein
by N-ethylmaleimide. Although P-glycoprotein contains
additional cysteine residues which might constitute male-
imide-binding sites, it has been demonstrated that both
Walker-A cysteines are accessible to covalent modification,
and that inhibition of ATPase activity is achieved by
derivatization of either cysteine [5,8,29]. It has been
observed that ATP incubation offered protection against
this N-ethylmaleimide-mediated inhibition, with between 2
and 10 m
M
nucleotide being necessary to restore ATPase
activity [8,49]. The protection of N-[
3
H]ethylmaleimide

labelling by preincubation with diverse nucleotides suggests
that ATP, 2-deoxy-ATP, p[NH]ppA and CTP can all bind
effectively at the NBDs of P-glycoprotein. This is consistent
with ATP and dATP being hydrolysis substrates with K
m
values approaching 1 m
M
[5,29]. It is also consistent with
p[NH]ppA being effective as an inhibitor of this hydrolysis
with an EC
50
of 0.4 m
M
[8,49]. Although we have demon-
strated that CTP is effective at preventing N-[
3
H]ethyl-
maleimide labelling, this nucleotide is not an effective
substrate for continuous hydrolysis [8,49], suggesting that its
binding does not induce the conformational changes that
accompany nucleotide hydrolysis.
Sequence and structural considerations
of P-glycoprotein NBDs
Our data do show some differences between the NBDs of
P-glycoprotein with respect to two nucleotides. The non-
hydrolysable analogue p[NH]ppA and the purine GTP
interact differentially. GTP fails to inhibit N-[
3
H]ethyl-
maleimide labelling of the N-terminal NBD, whereas

p[NH]ppA appears to have a lower affinity for the C-ter-
minal NBD. Can these differences be related to sequence and
structural properties of the two NBDs? To address this, we
have generated homology models for the N-terminal and
C-terminal NBDs of P-glycoprotein based on the crystal
structure of TAP1 [36] (Fig. 5). A representative model of
the N-terminal NBD is shown in Fig. 5A (the C-terminal
NBD model has a similar structure and therefore is not
shown). Figure 5B displays in detail the vicinity of the ATP
molecule demonstrating the exposure of the cysteine residue
(Cys431) to the ATP-binding pocket. Two sequence differ-
ences between the N-terminal and C-terminal NBD in this
region are highlighted in ball-and-stick fashion in Fig. 5B.
The first is the presence of an asparagine (Asn428) in the
Walker-A motif of the N-terminal NBD, which is replaced
by a serine (Ser1071) in the C-terminal NBD. The side chain
of this asparagine is less than 6 A
˚
from the Pb–Ob phospho-
anhydride bond of ATP. This is the bond that is altered in
p[NH]ppA, suggesting that replacement of Asn428 by
Ser1071 could confer subtle alterations on p[NH]ppA
binding, in agreement with the results obtained here. The
nonidentical interaction of GTP may be related to another
variation in sequence in the vicinity of the ATP-binding
pocket, specifically in the ABC-specific b-sheet subdomain
Ó FEBS 2003 Functional symmetry of P-glycoprotein NBDs (Eur. J. Biochem. 270) 1489
(Fig. 5A). Our homology models of P-glycoprotein NBDs
suggest that replacement of residue Ser400 in the N-terminal
NBD by the bulkier Asn1043 in the C-terminal NBD may be

sufficient to impart the observed effects on interaction with
GTP (possibly mediated via water molecules).
Functional and structural dissimilarity in the NBDs
of ABC transporters
For many eukaryotic ABC transporters, there are many
data to support functional asymmetry between the NBDs in
mediating transport. This includes demonstrations that this
functional asymmetry is a result of the inherent properties of
the NBDs, rather than of their interactions with the TMDs
(e.g [50] and see introduction for further references). Recent
structural determinations of both intact ABC transporters
and NBD proteins also lend weight to asymmetry in these
proteins. The crystal dimer of the NBD of the maltose-
uptake system of Thermococcus littoralis (MalK) consists of
two monomers with deviation from perfect twofold sym-
metry [44], although an analysis of how this might be related
to nucleotide binding is precluded by the presence of
pyrophosphate in the binding pocket, rather than ATP [44].
In addition, the cryo-electron microscopy structure of YvcC
supports structural asymmetry in ABC transporters [51].
YvcC is a homodimeric protein (each monomer consisting
of a single TMD and NBD). The structures identified as the
NBDs are of different dimensions and thus, presumably,
different conformations [51]. Whether this is attributable to
NBD–NBD interactions or NBD–TMD interactions awaits
a higher-resolution structure.
Summary
We have shown that the two NBDs of P-glycoprotein are
substantially functionally symmetrical in terms of their
binding to diverse nucleotides. Any functional asymmetry

observed in the intact transporter is probably not entirely
due to inherent properties of the NBD, and presumably
reflects either differences in the rate of hydrolysis or the
effects of interdomain interactions. In particular, our data
demonstrate that, in isolation, both NBDs interact identi-
cally with ATP, in agreement with a recent spectroscopic
study of P-glycoprotein, which observed secondary struc-
tural asymmetry as a result of nucleotide hydrolysis, but not
nucleotide binding [10]. Our resultant hypothesis that
either NBD may be recruited to hydrolyse ATP
during the transport cycle will be tested by further
experiments involving single-cysteine isoforms of full-length
P-glycoprotein.
Acknowledgements
This work was funded by a Wellcome Trust Career Development
Fellowship to I.D.K., a Wellcome Trust Vacation Scholarship to
J.A.W., and Cancer Research UK Grant to R.C. We thank Natalie
Gabriel for preliminary purifications of MBP-NBD fusion proteins,
and Georgios Samoilis for assistance with site-directed mutagenesis. We
thank Catherine Martin, Mark Gabriel, Alice Rothnie, Janet Storm,
andDrB.Nardfordiscussions.
References
1. Higgins, C.F. (1992) ABC transporters: from microorganisms to
man. Annu.Rev.CellBiol.8, 67–113.
Fig. 5. Homology modelling of P-glycoprotein NBDs. Structural
models for the N-terminal and C-terminal NBD of P-glycoprotein
were constructed as described in the text. (A) N-terminal NBD model
presented in ribbon format, with a-helices represented as spirals, and
a-strands as arrows. The ATP molecule is shown in ball-and-stick
format. The scale bar represents 5 A

˚
.(B)Aclose-upviewofthe
nucleotide-binding pocket in the N-terminal homology model. ATP is
shown as before, as are the side chains of Cys431, Asn428 and Ser400.
The scale bar represents 2 A
˚
.
1490 G. Berridge et al.(Eur. J. Biochem. 270) Ó FEBS 2003
2. Holland, I.B. & Blight, M.A. (1999) ABC-ATPases, adaptable
energy generators fuelling transmembrane movement of a variety
of molecules in organisms from bacteria to humans. J. Mol. Biol.
293, 381–399.
3. Kerr, I.D. (2002) Structure and association of ATP binding cas-
sette transporter nucleotide-binding domains. Biochim. Biophys.
Acta 1561, 47–64.
4. Urbatsch, I.L., Sankaran, B., Bhagat, S. & Senior, A.E. (1995)
Both P-glycoprotein nucleotide-binding sites are catalytically
active. J. Biol. Chem. 270, 26956–26961.
5. al Shawi, M.K. & Senior, A.E. (1993) Characterization of the
adenosine triphosphatase activity of Chinese hamster P-glyco-
protein. J. Biol. Chem. 268, 4197–4206.
6. Senior, A.E., al Shawi, M.K. & Urbatsch, I.L. (1995) The catalytic
cycle of P-glycoprotein. FEBS Lett. 377, 285–289.
7. Takada, Y., Yamada, K., Taguchi, Y., Kino, K., Matsuo, M.,
Tucker, S.J., Komano, T., Amachi, T. & Ueda, K. (1998) Non-
equivalent cooperation between the two nucleotide-binding folds
of P-glycoprotein. Biochim. Biophys. Acta 1373, 131–136.
8. Loo, T.W. & Clarke, D.M. (1995) Covalent modification of
human P-glycoprotein mutants containing a single cysteine in
either nucleotide-binding fold abolishes drug- stimulated ATPase

activity. J. Biol. Chem. 270, 22957–22961.
9. Hrycyna, C.A., Ramachandra, M., Germann, U.A., Cheng, P.W.,
Pastan, I. & Gottesman, M.M. (1999) Both ATP sites of human
P-glycoprotein and essential but not symmetric. Biochemistry 38,
13887–13899.
10. Vigano,C.,Julien,M.,Carrier,I.,Gros,P.&Ruysschaert,J.M.
(2002) Structural and functional asymmetry of the nucleotide-
binding domains of P-glycoprotein investigated by attenuated
total reflection Fourier transform infrared spectroscopy. J. Biol.
Chem. 277, 5008–5016.
11. Szakacs, G., Ozvegy, C., Bakos, E., Sarkadi, B. & Varadi, A.
(2001) Role of glycine-534 and glycine-1179 of human multidrug
resistance protein (MDR1) in drug-mediated control of ATP
hydrolysis. Biochem. J. 356, 71–75.
12. Urbatsch,I.L.,Gimi,K.,Wilke-Mounts,S.&Senior,A.E.(2000)
Investigation of the role of glutamine-471 and glutamine-1114 in
the two catalytic sites of P-glycoprotein. Biochemistry 39, 11921–
11927.
13. Urbatsch,I.L.,Gimi,K.,Wilke-Mounts,S.&Senior,A.E.(2000)
Conserved Walker A Ser residues in the catalytic sites of P-gly-
coprotein are critical for catalysis and involved primarily at the
transition state step. J. Biol. Chem. 275, 25031–25038.
14. Gao,M.,Cui,H.R.,Loe,D.W.,Grant,C.E.,Almquist,K.C.,
Cole, S.P. & Deeley, R.G. (2000) Comparison of the functional
characteristics of the nucleotide binding domains of multidrug
resistance protein 1. J. Biol. Chem. 275, 13098–13108.
15. Hou, Y., Cui, L., Riordan, J.R. & Chang, X. (2000) Allosteric
interactions between the two non-equivalent nucleotide binding
domains of multidrug resistance protein MRP1. J. Biol. Chem.
275, 20280–20287.

16. Lapinski, P.E., Neubig, R.R. & Raghavan, M. (2001) Walker A
lysine mutations of TAP1 and TAP2 interfere with peptide
translocation but not peptide binding. J. Biol. Chem. 276, 7526–
7533.
17. Saveanu, L., Daniel, S. & van Endert, P.M. (2001) Distinct
functions of the ATP binding cassettes of transporters associated
with antigen processing: a mutational analysis of Walker A and B
sequences. J. Biol. Chem. 276, 22107–22113.
18. Proff, C. & Kolling, R. (2001) Functional asymmetry of the two
nucleotide binding domains in the ABC transporter Ste6. Mol.
Gen. Genet. 264, 883–893.
19. Anderson, M.P. & Welsh, M.J. (1992) Regulation by ATP and
ADP of CFTR chloride channels that contain mutant nucleotide-
binding domains. Science 257, 1701–1704.
20. Carson, M.R., Travis, S.M. & Welsh, M.J. (1995) The two
nucleotide-binding domains of cystic fibrosis transmembrane
conductance regulator (CFTR) have distinct functions in con-
trolling channel activity. J. Biol. Chem. 270, 1711–1717.
21. Druley, T.E., Stein, W.D. & Roninson, I.B. (2001) Analysis of
MDR1 P-glycoprotein conformational changes in permeabilized
cells using differential immunoreactivity. Biochemistry 40, 4312–
4322.
22. Martin, C., Berridge, G., Mistry, P., Higgins, C.F., Charlton, P. &
Callaghan, R. (2000) Drug binding sites on P-glycoprotein are
altered by ATP binding prior to nucleotide hydrolysis. Biochem-
istry 39, 11901–11906.
23. Wang, G., Pincheira, R., Zhang, M. & Zhang, J T. (1997) Con-
formational changes of P-glycoprotein by nucleotide binding.
Biochem. J. 328, 897–904.
24. Rosenberg, M.F., Velarde, G., Ford, R.C., Martin, C., Berridge,

G., Kerr, I.D., Callaghan, R., Schmidlin, A., Wooding, C., Lin-
ton, K.J. & Higgins, C.F. (2001) Repacking of the transmembrane
domains of P-glycoprotein during the transport ATPase cycle.
EMBO J. 20, 5615–5625.
25. Karpowich, N., Martsinkevich, O., Millen, L., Yuan, Y.R., Dai,
P.L., MacVey, K., Thomas, P.J. & Hunt, J.F. (2001) Crystal
structures of the MJ1267 ATP-binding cassette reveal an induced-
fiteffectattheATPaseactivesiteofanABCtransporter.Structure
9, 571–586.
26. Yuan, Y.R., Blecker, S., Martsinkevich, O., Millen, L., Thomas,
P.J. & Hunt, J.F. (2001) The crystal structure of the MJ0796 ATP-
binding cassette: Implications for the structural consequences of
ATP hydrolysis in the active site of an ABC-transporter. J. Biol.
Chem. 276, 32313–32321.
27. Locher, K.P., Lee, A.T. & Rees, D.C. (2002) The E. coli BtuCD
structure: a framework for ABC transporter architecture and
mechanism. Science 296, 1091–1098.
28. Smith, P.C., Karpowich, N., Millen, L., Moody, J.E., Rosen, J.,
Thomas, P.J. & Hunt, J.F. (2002) ATP binding to the motor
domain from an ABC transporter drives formation of a nucleotide
sandwich dimer. Mol. Cell. 10, 139–149.
29. Urbatsch, I.L., al Shawi, M.K. & Senior, A.E. (1994) Character-
ization of the ATPase activity of purified Chinese hamster P-gly-
coprotein. Biochemistry 33, 7069–7076.
30. Hung, L.W., Wang, I.X., Nikaido, K., Liu, P.Q., Ames, G.F. &
Kim, S.H. (1998) Crystal structure of the ATP-binding subunit of
an ABC transporter. Nature (London) 396, 703–707.
31. Taylor, A.M., Storm, J., Linton, K.J., Gabriel, M., Blott,
E.J.,Martin,C.,Higgins,C.F.,Mistry,P.,Charlton,P.&
Callaghan, R. (2001) Subtle pharmacological differences

between a Cys-less variant of the human multidrug resistance
protein, P-glycoprotein, and wild-type. Br.J.Pharmacol.134,
1609–1618.
32. Hanahan, D. (1983) Studies on transformation of Escherichia coli
with plasmids. J. Mol. Biol. 166, 557–580.
33. Chifflet, S., Torriglia, A., Chiesa, R. & Tolosa, S. (1988) A method
for the determination of inorganic phosphate in the presence of
labile organic phosphate and high concentrations of protein:
application to lens ATPases. Anal. Biochem. 168,1–4.
34. Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994) CLUSTAL
W: improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–
4680.
35. Sali, A. & Blundell, T.L. (1993) Comparative protein
modelling by satisfaction of spatial restraints. J. Mol. Biol. 234,
779–815.
36. Gaudet, R. & Wiley, D.C. (2001) Structure of the ABC ATPase
domain of human TAP1, the transporter associated with antigen
processing. EMBO J. 20, 4964–4972.
Ó FEBS 2003 Functional symmetry of P-glycoprotein NBDs (Eur. J. Biochem. 270) 1491
37. Rodriguez, R., Chinea, G., Lopez, N., Pons, T. & Vriend, G.
(1998) Homology modeling, model and software evaluation: three
related resources. Bioinformatics 14, 523–528.
38. Kraulis, P.J. (1991) MOLSCRIPT: a program to produce both
detailed and schematic plots of protein structure. J. Appl. Crys-
tallogr. 24, 946–950.
39. Dayan, G., Jault, J.M., Baubichon Cortay, H., Baggetto, L.G.,
Renoir, J.M., Baulieu, E.E., Gros, P. & Di Pietro, A. (1997)
Binding of steroid modulators to recombinant cytosolic domain

from mouse P-glycoprotein in close proximity to the ATP site.
Biochemistry 36, 15208–15215.
40. de Wet, H., McIntosh, D.B., Conseil, G., Baubichon-Cortay, H.,
Krell, T., Jault, J.M., Daskiewicz, J.B., Barron, D. & Di Pietro, A.
(2001) Sequence requirements of the ATP-binding site within the
C-terminal nucleotide-binding domain of mouse P-glycoprotein:
structure-activity relationships for flavonoid binding. Biochemistry
40, 10382–10391.
41. Reference withdrawn.
42. Duplay, P., Bedouelle, H., Fowler, A., Zabin, I., Saurin, W. &
Hofnung, M. (1984) Sequences of the malE gene and of its pro-
duct, the maltose-binding protein of Escherichia coli K12. J. Biol.
Chem. 259, 10606–10613.
43. Chen,C.J.,Clark,D.,Ueda,K.,Pastan,I.,Gottesman,M.M.&
Roninson, I.B. (1990) Genomic organization of the human multi-
drug resistance (MDR1) gene and origin of P-glycoproteins.
J. Biol. Chem. 265, 506–514.
44. Diederichs, K., Diez, J., Greller, G., Muller, C., Breed, J., Schnell,
C.,Vonrhein,C.,Boos,W.&Welte,W.(2000)Crystalstructure
of MalK, the ATPase subunit of the trehalose/maltose ABC
transporter of the archaeon Thermococcus litoralis. EMBO J. 19,
5951–5961.
45. Hopfner, K.P., Karcher, A., Shin, D.S., Craig, L., Arthur, L.M.,
Carney, J.P. & Tainer, J.A. (2000) Structural biology of Rad50
ATPase: ATP-driven conformational control in DNA double-
strand break repair and the ABC-ATPase superfamily. Cell 101,
789–800.
46. Brewer, C. & Riehm, J. (1967) Evidence for possible non-specific
reactions between N-ethylmaleimide and proteins. Anal. Biochem.
18, 248–255.

47. Wang, C., Castro, A.F., Wilkes, D.M. & Altenberg, G.A. (1999)
Expression and purification of the first nucleotide-binding domain
and linker region of human multidrug resistance gene product:
comparison of fusions to glutathione S-transferase, thioredoxin
and maltose-binding protein. Biochem. J. 338, 77–81.
48. Wilkes, D.M., Wang, C., Aristimuno, P.C., Castro, A.F. &
Altenberg, G.A. (2002) Nucleotide triphosphatase activity of
the N-terminal nucleotide-binding domains of the multidrug
resistance proteins P-glycoprotein and MRP1. Biochem. Biophys.
Res. Commun. 296, 388–394.
49. al Shawi, M.K., Urbatsch, I.L. & Senior, A.E. (1994) Covalent
inhibitors of P-glycoprotein ATPase activity. J. Biol. Chem. 269,
8986–8992.
50. Daumke, O. & Knittler, M.R. (2001) Functional asymmetry of the
ATP-binding-cassettes of the ABC transporter TAP is determined
by intrinsic properties of the nucleotide binding domains. Eur. J.
Biochem. 268, 4776–4786.
51. Chami, M., Steinfels, E., Orelle, C., Jault, J.M., Di Pietro, A.,
Rigaud, J.L. & Marco, S. (2002) Three-dimensional structure by
cryo-electron microscopy of YvcC, an homodimeric ATP-binding
cassette transporter from Bacillus subtilis. J. Mol. Biol. 315, 1075–
1085.
1492 G. Berridge et al.(Eur. J. Biochem. 270) Ó FEBS 2003