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

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-ter

The nucleotide-binding domains of P-glycoprotein

labelling

Georgina Berridge1, Jennifer A Walker1, Richard Callaghan1and Ian D Kerr1,2

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],

and that hydrolysis at the two NBDs may occur in an

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: ian.kerr@nottingham.ac.uk

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)

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-[3H]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¢-GAAGAGTGGGCAACGGATCCGAT AATATTTAAG and 5¢-CATTTCCTGCTGTCTGCAG 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¢-GGCAGCAGTGGCTGTGG 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¢-CAGCACGGAAGGCGAATTCCCG AACACATTG and 5¢-CTTTGTTCCAGCCTGCAGT CAGACCATTGAAAA, respectively (restriction cloning sites underlined) N-terminal and C-terminal NBDs were cloned into the pMal-C2X vector at the BamHI/PstI and 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)1ampicillin and grown overnight at

37C with shaking at 200 r.p.m., and then diluted 1 : 80 into

400 mL Luria–Bertani broth/ampicillin Growth was con-tinued at 37C until an A600of 0.5 was achieved Cultures were then cooled to 25C and induced with 0.2 mM

isopropyl thio-b-D-galactoside After 3–4 h shaking at

25C, bacteria were harvested by centrifugation at 3000 g Purification of fusion proteins

Bacterial cell pellets were resuspended in 10 mL lysis buffer (50 mMTris/HCl, 150 mMNaCl, 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 4C (Beckman TLA 100 rotor) Soluble protein was diluted to a concentration of 3 mgÆmL)1and 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 lM maltose Bound fusion proteins were eluted by subsequent washes of 2 mL lysis buffer containing 1 mM 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]

Labelling withN-ethylmaleimide

N-[3H]Ethylmaleimide (specific radioactivity 40–60 CiÆ

mmol)1, 1 mCiÆmL)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-[3H]Ethylmaleimide (4 mCiÆmL)1) was then

frozen in aliquots at)20 C N-[3H]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-[3H]ethylmaleimide

(0.6 lM, determined by liquid-scintillation counting) for

increasing times at 22C 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 AMPLIFY

(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-[3H]ethylmaleimide labelling, 1.5 lg protein in lysis

buffer, supplemented with 5 mM MgCl2, was incubated

with increasing concentrations of nucleotide at 37C for

30 min N-[3H]Ethylmaleimide was then added to a final

concentration of 0.6 lMand incubated at 22C 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-[3H]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 usingPRISM(GraphPad, San Diego, CA, USA)

to the equation:

y¼ ymax ð1  expktÞ

where y¼ percentage saturation, t ¼ time, k ¼ time

con-stant for labelling (T0.5¼ 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:

y¼ bottom þ ðtop  bottomÞ

1þ 10ðlog IC 50log x Þn

where y¼ percentage of reference signal, x ¼ nucleotide concentration, bottom¼ minimum labelling inten-sity, top¼ saturation of labelling, IC50¼ concentration required to reduce labelling intensity to 50% of its maximum, and n¼ 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 T0.5 and IC50 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 usingCLUSTALW [34] A series of 10 homology models of each NBD was constructed, using the program MODEL-LER_6V2 [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 usingMOLSCRIPT[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

mutagenesis Thus, both 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

toN-ethylmaleimide The initial experiments provided a detailed characterization

of the binding of N-[3H]ethylmaleimide to MBP-NBD fusion proteins Time, temperature and concentration dependence of N-[3H]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, 22C 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-[3H]ethylmaleimide investigates the time course of deri-vatization of the cysteine residue in the ATP-binding pocket and demonstrates its accessibility to N-[3H]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 (T0.5) for the association of N-[3H]ethylmaleimide with MBP-NBD-Nter of 30.6 ± 1.5 min (n¼ 4), and with MBP-NBD-Cter

of 38.0 ± 4.3 min (n¼ 4) Unpaired t test demonstrates no difference in the T0.5for N-[3H]ethylmaleimide labelling of the two fusion proteins under these conditions

Preincubation with ATP but not ADP prevents N-[3H]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-[3H]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-[3H]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.

adding 0.6 lM N-[3H]ethylmaleimide This reaction was

allowed to proceed for 15 min at 22C, over which time any

inhibition of N-[3H]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-[3H]ethylmaleimide labelling are due to nucleotide

bind-ing only Nucleotide concentrations of up to 3.5 mMwere

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 mM was able to completely

prevent N-ethylmaleimide labelling under the reaction

conditions used The mean data, obtained in at least eight

experiments, return IC values for the inhibition of

N-[3H]ethylmaleimide labelling for MBP-NBD-Nter of 1.8 ± 0.2 mM(n¼ 8) and for MBP-NBD-Cter of 2.3 ± 0.2 mM(n¼ 9) The difference in the potency of reduction

in N-[3H]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-[3H]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-[3H]ethylmaleimide labelling (Table 1), with negligible displacement at concentrations up to 3.5 mMin 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-strated at concentration of up to 3.5 mMADP (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.

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-[3H]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 (IC50¼ 2.2 ± 0.2 mM) and the C-terminal

NBD (IC50¼ 2.3 ± 0.3 mM) Neither dTTP nor ITP was

able to fully prevent N-[3H]ethylmaleimide labelling by

preincubation at concentrations as high as 3.5 mM

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-[3H]ethylmaleimide

labelling between the two NBDs was observed with

p[NH]ppA and GTP GTP preincubation, at

concentra-tions up to 3.5 mM 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 mM 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-[3H]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 (IC50¼ 0.9 ± 0.1 mM), 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-[3H]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-[3H]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.

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

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-[3H]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-[3H]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.

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

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-[3H]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-[3H]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-[32P]ATP labelling to determine relative affinities of

nucleotides However, the maximum concentration of

commercially available 8-azido-[32P]ATP is only 100 lMin

methanol Avoiding excess solvent in labelling experiments

would impose an upper limit on the achievable

concentra-tion of 8-azido-[32P]ATP of 5 lM This is two orders of

magnitude below the Kmfor 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 mM nucleotide being necessary to restore ATPase activity [8,49] The protection of N-[3H]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 Km values approaching 1 mM [5,29] It is also consistent with p[NH]ppA being effective as an inhibitor of this hydrolysis with an EC50of 0.4 mM[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

(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, and Dr B Nard for discussions.

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