Báo cáo khoa học: Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1 pot

Transmembrane helix 12 plays a pivotal role in coupling
energy provision and drug binding in ABCB1
Emily Crowley1, Megan L. O’Mara2, Ian D. Kerr3 and Richard Callaghan1
1 Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, UK
2 Molecular Dynamics Group, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia
3 School of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, UK

Keywords
ABC transporter; bioenergetic coupling; drug
resistance; efflux pumps; P-glycoprotein
Correspondence
R. Callaghan, Nuffield Department of Clinical
Laboratory Sciences, John Radcliffe
Hospital, University of Oxford, Oxford, OX3
9DU, UK
Fax: +44 1865 221 834
Tel: +44 1865 221 110
E-mail:
(Received 5 May 2010, revised 2 July 2010,
accepted 27 July 2010)
doi:10.1111/j.1742-4658.2010.07789.x

Describing the molecular details of the multidrug efflux process of ABCB1,
in particular the interdomain communication associated with bioenergetic
coupling, continues to prove difficult. A number of investigations to date
have implicated transmembrane helix 12 (TM12) in mediating communication between the transmembrane domains and nucleotide-binding domains
(NBDs) of ABCB1. The present investigation further addressed the role of
TM12 in ABCB1 by characterizing its topography during the multidrug
efflux process with the use of cysteine-directed mutagenesis. Cysteines were
introduced at various positions along TM12 and assessed for their ability
to covalently bind thiol-reactive fluorescent probes with differing physiochemical properties. By analysing each isoform in the basal, ATP-bound

and posthydrolytic states, it was possible to determine how the local environment of TM12 alters during the catalytic cycle. Labelling with hydrophobic CM and zwitterionic BM was extensive throughout the helix in the
basal, prehydrolytic and posthydrolytic states, suggesting that TM12 is in a
predominantly hydrophobic environment. Overall, the carboxy region
(intracellular half) of TM12 appeared to be more responsive to changes in
the catalytic state of the protein than the amino region (extracellular half).
Thus, the carboxy region of TM12 is suggested to be responsive to nucleotide binding and hydrolysis at the NBDs and therefore directly involved in
interdomain communication. This data can be reconciled with an atomicscale model of human ABCB1. Taken together, these results indicate that
TM12 plays a key role in the progression of the ATP hydrolytic cycle in
ABCB1 and, in particular, in coordinating conformational changes between
the NBDs and transmembrane domains.

Introduction
ABCB1 (P-glycoprotein) is a member of the ATP-binding cassette (ABC) family of membrane transporters,
and is located in the plasma membrane of cells. The
transporter is localized to a number of tissues associated with absorptive, secretory or barrier roles [1–3],

and its primary function is therefore to provide a
defensive mechanism against xenobiotics. ABCB1 provides this defence by acting as a multidrug efflux
pump. The expression pattern in physiological tissues
enables ABCB1 to play a prominent role in shaping

Abbreviations
ABC, ATP-binding cassette; AMP-PNP, 5¢-adenylylimidodiphosphate; BM, BODIPY maleimide; CM, coumarin maleimide; FM, fluorescein
maleimide; Lext, maximum extent of labelling; NBD, nucleotide-binding domain; TMD, transmembrane domain; TM6, transmembrane helix 6;
TM12, transmembrane helix 12.

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E. Crowley et al.

the pharmacokinetic profile (adsorption, distribution,
metabolism and excretion) of many commonly used
medications [4]. Unfortunately, cancer cells overexpress
ABCB1 in order to evade the toxic effects of anticancer drugs, a phenomenon known as multidrug resistance. The extraordinary range of compounds
recognized by ABCB1 (over 200 known drugs) makes
it a powerful mediator of resistance against chemotherapeutic intervention in a number of cancer types. The
ability to recognize such an array of compounds
remains a biological enigma, thereby making the development of inhibitors that may restore the efficacy of
chemotherapy in cancer treatment a difficult task.
The functional unit of ABCB1 consists of two transmembrane domains (TMDs), each comprising six
membrane-spanning helices, and two nucleotide-binding domains (NBDs) [5]. Much is understood regarding NBD function, owing to the high sequence
homology between members of the ABC transporter
family. Furthermore, several crystal structures of ABC
transporters have been solved in the presence and
absence of nucleotides, improving our understanding
of the mechanism of transport [6–9]. However, much
remains unclear about structure–function relationships
of the TMDs of multidrug resistance pumps, including
the location of the drug-binding sites and the molecular mechanism underlying drug translocation. The
˚
recent 4–4.3 A resolution crystal structure of fulllength ABCB1 has provided a location for the binding
of a purpose-built peptide inhibitor [6]. However, more
pharmacological information is required to evaluate
this inhibitor and how its binding relates to more
established substrates or modulators [10–12].
Another unresolved issue pertaining to ABCB1 function is the molecular detail of the process of coupling
between the NBDs and TMDs. The most striking evidence for the presence of coupling between the two

domains is the ability of transported drugs to stimulate
the basal rate of ATP hydrolysis by ABCB1 [13,14]. It
is well established that drug binding occurs in the
TMDs, and stimulation of hydrolysis therefore
requires long-distance communication with the cytosolic NBDs. This is supported by evidence that mutations of numerous residues within the TMDs are
capable of disrupting the stimulation of ATP hydrolysis [15–19]. Moreover, drug translocation and ATP
hydrolysis must be coordinated for active efflux. This
requires interdomain communication in both the TMD
to NBD and NBD to TMD directions [20]. The latter
route has also been demonstrated; for example, binding of the nucleotide analogues ATPcS or 5¢-adenylylimidodiphosphate (AMP-PNP) to the NBDs of ABCB1
was shown to significantly decrease the binding of the

Role of TM12 in bioenergetic coupling in ABCB1

UIC2 antibody, which recognizes a conformation-sensitive epitope in the TMD [21,22]. Furthermore, the
cryo-electron microscopy structure of ABCB1 showed
that in the presence of the AMP-PNP the architecture
of the TMDs is significantly rearranged [23,24].
Together, these experiments demonstrated that global conformational changes occur in the protein and
are relayed from the NBDs to the TMDs as a consequence of nucleotide binding and drug binding, respectively, thereby enabling active drug efflux by ABCB1.
However, we are yet to understand exactly how these
conformational changes are relayed between the TMD
and NBD, and how they enable drug translocation.
Transmembrane helix 6 (TM6) and transmembrane
helix 12 (TM12) are likely candidates to effect coupling,
given their direct links to the two NBDs of ABCB1.
We have previously constructed, and analysed, a series of TM6s with single cysteine mutations, and demonstrated that this helix plays a prominent role in the
coupling process in ABCB1 [25–28]. A number of mutations in TM6 caused alterations in drug-stimulated ATP
hydrolysis, irrespective of whether they contributed to
drug binding. Moreover, several residues in TM6 were

demonstrated to undergo topographical alterations during conformational changes of ABCB1. In a recent
study, we demonstrated, using a similar approach, that
the mutation of several residues within TM12 also influences the communication between the TMDs and
NBDs [15]. The present article describes the conformational changes adopted by TM12 in response to events
occurring in the NBDs. The data indicate that nucleotide binding and hydrolysis at the NBDs causes conformational changes that are transmitted through TM12.

Results
Three thiol-reactive fluorescent probes were used to
assess the relative accessibility of selected residues in
TM12 that had been mutated to cysteine. The probes
possess distinct physicochemical properties and have
been shown to partition to hydrophilic or hydrophobic
environments [25]. By assessment of the ability of each
probe to label residues in TM12, a topographical map
of the helix can be generated. Furthermore, trapping the
protein at distinct stages of the catalytic cycle will reflect
how the environment of individual residues in TM12
changes as ABCB1 switches conformational states.
The maximum extent of labelling of TM12
residues in ABCB1
The maximum extent of labelling (Lext) of selected
TM12 mutant isoforms was initially investigated with

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E. Crowley et al.

reconstituted protein in the basal (nucleotide-free)
state. Following incubation with the fluorescent probe,
the proteins were resolved by SDS ⁄ PAGE, and the
covalent binding of the probe was detected under UV
light. Figure 1 (lower panel) shows a representative
labelling reaction, in this case a time course for the
V988C isoform with coumarin maleimide (CM). The
gel in the upper panel of Fig. 1 shows the same gel but
stained with PageBlue to demonstrate purity of the
samples and to enable normalization of labelling for
protein loading. Labelling was time dependent during
the 300 min incubation, and the extents of labelling
were quantified in comparison with that found with
cysteine-less ABCB1 and the G324C isoform. The
G324C mutant was assigned as the positive control
and given a value of 100%, as this residue is located
on an external loop and is freely accessible to each of
the probes used [25,28]. Furthermore, the complete

150 kDa

100 kDa

(i)

(ii)

(iii) (iv) (v) (vi) (vii)

150 kDa
100 kDa

Fig. 1. Detection of CM labelling of the V988C isoform. SDS ⁄ PAGE
analysis of the V988C isoform incubated in the presence of CM for
0–300 min. The reaction was stopped at various time points by the
addition of dithiothreitol. Upper panel: the gel protein was visualized
with PageBlue staining to indicate sample purity and to enable loading correction. Lower panel: the samples were resolved by
SDS ⁄ PAGE and the protein was visualized with the BioDocIt system, using a UV light source. Molecular mass markers are shown
on the left. Lane assignments are: (i) 300 min; (ii) 120 min; (iii)
60 min; (iv) 30 min; (v) 10 min; and (vi) 0 min. Lane (vii) contains
the G324C isoform, which has been assigned a 100% value for
labelling with BM.

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labelling of the G324C mutant with the zwitterionic
and hydrophilic probes BODIPY maleimide (BM) and
fluorescein maleimide (FM), respectively, demonstrated
that the protein was not preferentially oriented in one
direction within the proteoliposomes. Consequently,
labelling of TM12 isoforms was determined as a
percentage of G324C labelling, as outlined in Experimental procedures. Additionally, labelling of the cysteine-less ABCB1 isoform was also examined as a
negative control. Any nonspecific association of the
three probes with cysteine-less ABCB1 was subtracted
from the specific labelling intensity observed with the
single-cysteine-containing isoforms. Obtaining full
labelling and its accurate quantitation are difficult to
achieve in practice, resulting in occasional instances

where values for the Lext of single-cysteine isoforms
are apparently > 100%. The approach does, however,
provide strong predictions of relative labelling propensity, reflecting accessibility of the specified residue.
Labelling of each isoform was analysed by densitometry and plotted as a function of time, as shown for
the M986C isoform for the three probes in Fig. 2A.
Nonlinear regression of the exponential reaction curve
estimated that the maximum extent of labelling for the
representative curve of the M986C isoform in the basal
state was 78% for CM (t1 ⁄ 2 = 8 min), 59% for BM
(t1 ⁄ 2 = 4 min) and 23% for FM (t1 ⁄ 2 = 45 min).
Clearly, this mutant isoform was avidly labelled with
the hydrophobic (CM) and zwitterionic (BM) probes,
on the basis of the extent and rapid half-life of the
interactions. In contrast, the hydrophilic FM displayed
only partial labelling, with a considerably longer halftime for the reaction. Similar analysis was undertaken
for each of the TM12 single-cysteine mutants (using
multiple protein preparations) in the basal (i.e. nucleotide-free) state; the extent and time course of labelling
are shown in Table 1.
All of the mutant isoforms examined were capable
of interacting with CM, which has a high octanol ⁄ water partition coefficient, indicating a preference
for hydrophobic regions. The central region of TM12,
from V982C to M986C, displayed the highest extent of
labelling, with Lext values of 75–100%. The C-terminal
stretch (V988C–F994C) was also capable of interacting
with CM, albeit with lower values of Lext, in the range
50–60%. The lowest labelling observed in the selection
of TM12 mutant isoforms was at L976C, with an Lext
of 38 ± 5%. Lower labelling presumably reflects the
location of the residues at the membrane–water interface or significant local steric hindrance. The half-lives
for the interaction of CM with ABCB1 ranged from

18 to 30 min, but did not reveal further details concerning the accessibility of the residues. Of the three

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E. Crowley et al.

Fig. 2. Analysis of probe labelling of mutant TM12 isoforms of
ABCB1. For each of the mutant isoforms, densitometric analysis
was used to quantify the UV images and values of labelling at each
time point. These were then expressed as a percentage of the
maximal extent of G324C labelling. The degree of labelling (% of
G324C level) was plotted as a function of time (min) and fitted with
an exponential reaction curve, using nonlinear least squares regression. (A) Representative data for labelling of the M986C isoform
with CM ( ), FM (d) and BM (s). (B) Representative data for labelling of the F994C isoform with FM in the basal (d), AMP-PNP (s)
and vanadate-trapped ( ) conformational states.

probes used in this investigation, the lipophilic CM
has the lowest molecular volume, and the interaction
of all but one residue at > 50% suggests that the helix
is in a hydrophobic environment.
BM also displays a high octanol ⁄ water partition
coefficient, and is therefore likely to reveal hydrophobic regions of ABCB1. However, unlike CM, this
probe contains a delocalized charge and is zwitterionic
in nature. Presumably, it assumes a more polarized orientation to accommodate this ampiphilicity. Like CM,
each of the TM12 residues examined was able to
undergo covalent modification with BM (Table 1),
which also suggests that the helix is located in a hydrophobic environment. A similar stretch of TM12
(namely V982C–V988C) displayed the greatest propensity to be labelled with BM, with only isoform M986C
being not completely labelled by the probe. Either side

of this central region was labelled with BM, but to
only a partial extent. Unlike the case for CM, there

Role of TM12 in bioenergetic coupling in ABCB1

was considerable variation in the half-lives of labelling
with BM of the TM12 mutant isoforms. The rate of
labelling (i.e. t1 ⁄ 2) was divided into fast (L986C–
G992C, average t1 ⁄ 2 $ 8 min) and slow (L976C–
G984C, average t1 ⁄ 2 $ 25 min) kinetics between the
carboxy-half and the amino-half, respectively. So,
although the helix is in a predominantly hydrophobic
region, there were some differences in topography
detected by the amphiphilic BM. This may suggest that
the carboxy region (i.e. cytosolic) lies at an interface
with a more hydrophilic domain of ABCB1, as this
region displayed more rapid labelling kinetics. This
hypothesis is supported by the fact that F994C, which
is proximal to the membrane surface, has a considerably greater Lext (111 ± 35%) for BM than the near
neighbours examined. An alternative explanation for
the two distinct kinetic divisions is that the amino
region is closely packed with another helix of ABCB1
that imparts steric restrictions on the kinetics of labelling in TM12.
The final probe used to examine the topography of
TM12 was the large hydrophilic FM; the extents and
time courses of interactions are shown in Table 1. The
data on extent of labelling data are in broad agreement
with the information provided by BM and CM. Only
one residue displayed avid labelling with FM, namely
F994C (Lext of 129 ± 24%), and this is at the extreme

carboxy-end of TM12, in proximity to the aqueous
environment. The proximally located S992C was also
able to interact with FM, although to only a partial
degree. The central and amino regions of TM12 displayed low labelling with the hydrophilic probe. However, two residues (G984C and M986C) in the central
region of TM12 did display labelling above background, albeit with Lext values of approximately 20%.
This may reflect that these two residues, although in a
hydrophobic local environment, are in the vicinity of a
more hydrophilic region of ABCB1. The rapid kinetics
of labelling of M986C with both BM and FM would
also support this local increase in hydrophilicity. It is
also worth noting that the extent of labelling is
affected by numerous factors, including steric effects
and local chemistry. These may have differential effects
on the kinetic parameters for certain residues.
Do conformational transitions alter the labelling
of residues in TM12 of ABCB1?
During the drug translocation process, ABCB1 adopts
a number of conformational states. As drug translocation is coupled to ATP hydrolysis, the conformational
transitions will be driven by events at the NBDs. If
TM12 is involved in the coupling process between the

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E. Crowley et al.

Table 1. Propensity for and rate of labelling of ABCB1 with thiol-reactive probes. The propensity for labelling of the TM12 mutant isoforms
was determined for the thiol-reactive probes CM, BM and FM. The reaction was stopped by the addition of dithiothreitol, and proteins were
resolved by SDS ⁄ PAGE. Densitometric analysis was used to determine the amount of labelling for each ABCB1 isoform. The extent (Lext)
and half-life (t1 ⁄ 2) of labelling were determined by nonlinear regression of the exponential reaction curve. The Lext for labelling is expressed
as the fraction of specific labelling of single-cysteine isoforms over the specific labelling of the G324C positive control. Values represent the
means ± standard errors of the mean from at least four independent protein preparations. –, no labelling; ND, values where the extent of
labelling was too low to accurately assign a value for t1 ⁄ 2.
CM

BM

FM

Mutant

Lext (%)

t1 ⁄ 2 (min)

Lext (%)

t1 ⁄ 2 (min)

Lext (%)

t1 ⁄ 2 (min)

L976C
A980C
V982C

G984C
M986C
V988C
G989C
S992C
F994C

38
53
98
73
89
53
64
55
51

29
34
15
29
25
37
15
22
11

66
54
164

84
51
221
21
51
111

29
20
27
22
3
18
9
4
13

–
–
–
13 ±
21 ±
–
–
32 ±
129 ±

–
–
–

ND
ND
–
–
25 ± 5
8±3

±
±
±
±
±
±
±
±
±

5
6
14
14
30
6
7
4
10

±
±
±

±
±
±
±
±
±

12
1
6
6
10
18
6
6
9

TMDs and NBDs, then it will presumably undergo
multiple topographical transitions during the catalytic
cycle. The previous section outlined the overall topography of TM12, by examining the accessibility of
introduced cysteines to maleimide-containing probes.
The next phase of investigation involved trapping
ABCB1 mutant isoforms at distinct conformational
stages (e.g. nucleotide-bound and immediately posthydrolysis) and reassessing the accessibility to maleimide
probes. The data thereby identified the dynamic
changes produced during transition between various
stages of the catalytic cycle.
The data in Fig. 2B show a representative time
course for labelling of the F994C mutant isoform with
FM in the basal, nucleotide-bound and vanadatetrapped conformations. The nucleotide-bound (prehydrolytic) conformation was achieved by incubation of

the mutant isoforms with the nonhydrolysable ATP
analogue AMP-PNP, as previously described [24]. The
posthydrolytic (but pre-ADP or phosphate release)
stage was produced by the vanadate-trapping procedure [24]. In the basal state, the protein was fully
labelled with FM (Lext of 105%); however, Lext was
reduced to 47% upon binding of AMP-PNP, and further reduced to 14% following vanadate trapping.
Accessibility data, as shown in Fig. 2B, were
obtained (using multiple protein preparations) for each
mutant isoform in the three conformations (nucleotidefree, AMP-PNP-bound and vanadate-trapped). Experiments were carried out as described in the previous
section, and the Lext and t1 ⁄ 2 parameters were obtained
from the labelling time course profiles. To simplify
analysis, a qualitative representation has been adopted
(Table 2).
3978

±
±
±
±
±
±
±
±
±

14
8
50
24
5

63
3
5
35

±
±
±
±
±
±
±
±
±

18
9
17
7
2
12
2
1
10

10
2

3
24

Conformational changes – amino region of TM12
As shown in Table 2, the amino region of TM12
(L976C–V982C) was not associated with large alterations in topography. In particular, accessibility of the
two residues to FM was negligible in the basal state,
and this did not change for the nucleotide-bound and
posthydrolytic states. There were, however, some subtle changes in accessibility of the two more hydrophobic probes. For example, L976C became less accessible
to BM, but more accessible to CM, following a shift
from the basal to the nucleotide-bound conformation.
As ABCB1 shifted to the posthydrolytic conformation,
the extent of BM labelling returned to the basal level,
whereas CM accessibility was retained. A980C shifted
to a low level of BM accessibility following nucleotide
binding by ABCB1, and again, an opposite shift was
seen for CM. The subsequent transition to a vanadatetrapped state resulted in the highest possible extent of
labelling for BM, but with no alteration for CM. Overall, nucleotide binding shifts the amino region to a distinctly hydrophobic environment, such that labelling
with the zwitterionic BM is, in fact, reduced. Given
that BM is ampiphilic, this would suggest a shift from
a possible interfacial region to a buried hydrophobic
one. Furthermore, the progression to the posthydrolytic state restored the topographical features seen under
basal conditions. In complete contrast, V982C did not
undergo any alterations of probe accessibility during
transition to the nucleotide-bound and posthydrolytic
conformational states. This was the only residue examined in TM12 that retained an unaltered topography
between the states despite the conformational changes
within the TMDs induced by the NBDs.

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E. Crowley et al.

Role of TM12 in bioenergetic coupling in ABCB1

Table 2. Relative accessibilities of TM12 residues. Accessibilities
of cysteines to FM, BM and CM were determined at distinct
stages of the catalytic cycle for each ABCB1 isoform. The extent of
labelling was compared with that of the cysteine-less ABCB1 isoform. Basal refers to the nucleotide-free state, whereas the AMPPNP and Vi-trapped states mimic prehydrolytic and posthydrolytic
states of the protein, respectively. +++, complete labelling
(Lext > 75%); ++, partial labelling (Lext = 50–75%); +, weak labelling
(Lext < 50%); ), labelling below the amount observed for cysteineless ABCB1. All values were determined as described in Table 1
and obtained from four independent protein preparations.

ABCB1 isoform
L976C

A980C

V982C

G984C

M986C

V988C

G989C

S992C

F994C

Catalytic
intermediate

CM

BM

FM

Basal
AMP-PNP
Vi trapped
Basal
AMP-PNP
Vi trapped
Basal
AMP-PNP
Vi trapped
Basal
AMP-PNP
Vi trapped
Basal
AMP-PNP
Vi trapped
Basal
AMP-PNP
Vi trapped
Basal

AMP-PNP
Vi trapped
Basal
AMP-PNP
Vi trapped
Basal
AMP-PNP
Vi trapped

++
+++
+++
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
++
+++
++
+++
+++
++
++
++

++
+++
++
++
++
+++

+++
++
+++
++
+
+++
+++
+++
+++
+++
+++
++
++
+++
++
+++
+++
+++
+
++
+
++
+++

++
+++
+++
+++

)
)
)
)
)
)
)
)
)
+
+
)
+
++
)
)
)
)
)
)
)
+
++
+
+++

++
+

Conformational changes – central region
Two of the residues examined in the central region
(G984C and M986C) of TM12 have been shown to
accommodate partial labelling with FM, suggestive of
aqueous accessibility in the basal state. At M986C, the
extent of labelling with the hydrophilic probe was
increased following the addition of nonhydrolysable
nucleotide. This was accompanied by a moderate
increase in labelling with the zwitterionic BM, but with
a reduction in accessibility with the hydrophobic CM.
This pattern of change suggests a shift towards a more
polar environment for this central residue. This
appeared to be a transient shift in microenvironment,
as the posthydrolytic state adopted a topography

similar to that in the basal configuration. G984C
underwent a broadly similar shift in topography as
M986C, although the degree of alteration was somewhat less striking.
Conformational changes – proximal to the central
region
The region immediately proximal to the centre of
TM12 (V988C–G989C) showed avid labelling by both
of the lipophilic probes (BM and CM) in the basal
configurations, and there were no significant alterations in accessibility upon progression of the catalytic
cycle. Labelling of V988C and G989C with the hydrophilic FM was negligible, regardless of the conformational state. The refractoriness of labelling to
conformational change is clearly demonstrated by
G989C. In particular, this residue displayed the lowest

overall accessibility to covalent modification, regardless
of the conformational state. At no stage of the catalytic cycle was either CM or BM able to fully label
G989C, which was the only residue to exhibit this
property. Similarly, no interaction between the hydrophilic FM and G989C was observed. The variation in
physicochemical properties of the three probes suggests
that the inherently low labelling at any stage of the
catalytic cycle was unlikely to result from the local solvent environment. A more likely explanation is steric
hindrance to labelling by neighbouring residues or helices in the TMD. The labelling properties of V988C–
G989C suggest that this region of TM12 undergoes
minimal conformational transition.
Conformational changes – carboxy region
Considerably greater changes in accessibility to probes
were observed at the extreme carboxy region of TM12,
suggesting a more prominent role in mediating conformational transitions. In the basal state, none of the
probes could effect complete labelling of the S992C
isoform. However, progression to the nucleotide-bound
state resulted in a universal increase in accessibility of
the residue to covalent modification by all three
probes. Further progression to the posthydrolytic state
caused a reversion in accessibility in comparison to
that seen in the basal state. The uniform changes in
accessibility to three probes with distinct chemical
properties suggest that the adoption of the nucleotidebound state relieves the steric hindrance to labelling
found in the basal conformation, and that this is
restored as the catalytic cycle continues.
F994C displays the highest accessibility of any residue in the basal conformation of ABCB1, which may

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reflect localization at the membrane–solute interface.
There was no alteration in the extent of labelling by
BM in any conformational state examined. In contrast,
there was a dramatic reduction in labelling by the
hydrophilic FM as the protein progressed to the nucleotide-bound and posthydrolytic states. This was
accompanied by a concomitant increase in accessibility
to the hydrophobic CM. Clearly, F994C undergoes
considerable changes in accessibility, suggestive of
a move from a relatively hydrophilic region to a
more lipophilic one as ABCB1 binds and hydrolyses
nucleotide.

Discussion
TM12 has previously been demonstrated to play an
integral role in coupling between the drug binding and
translocation process (TMD), with the hydrolysis of
nucleotide (NBD) [15,29]. Moreover, perturbation of
TM12 altered not only drug-stimulated ATP hydrolysis, but also the inherent (basal) hydrolytic activity.
The latter demonstrates that activity of the NBDs,
even in the absence of substrate, is subject to some
degree of control or modulation by the TMDs of
ABCB1. TM6 in the amino-half of ABCB1 has often
been regarded as a mirror image of TM12, but, from a
purely functional perspective, cysteine introduction

within TM12 generated considerably greater functional
consequences for ABCB1 than corresponding mutations in TM6. The present study investigated whether
the ‘mirror image’ relationship holds true, particularly
with respect to the topographical changes in TM12
throughout the catalytic cycle.
The topographical changes were examined by introducing cysteines at distinct positions in TM12 and
assessing their accessibility to covalent modification
with thiol-reactive probes. In order to determine how
changes in the extent and rate of labelling reflect conformational changes in TM12, we used molecular
models of ABCB1 [30] in the basal and ATP-bound
states as the basis for in silico characterization. Homology modelling has previously been used to characterize
the effects of mutations in TM12 ⁄ TM6 on the overall
function of ABCB1 and to interpret the changes in
labelling accessibility that occur in TM6 [27]. This
approach provided a mechanistic explanation for the
role of TM6 in the translocation mechanism of
ABCB1, and was reproduced in the present investigation for TM12.
The changes in the accessibility to probes of mutated
residues within TM12 showed both increases and
decreases in the propensity for labelling throughout
the catalytic states, suggesting that TM12 undergoes
3980

conformational alterations, or is subjected to changes
in its local environment. There were two major observations to be drawn from studying the topography of
TM12 in the homology model. First, the midregion of
the helix, i.e. V982–G984, was rigid with respect to the
intracellular and extracellular sections of the helix in
the basal and ATP-bound states of the model, and that
this section of TM12 appeared to act as an anchor

around which the rest of the helix moved. Second, the
model shows that the intracellular part of TM12 also
contributes residues (between Met986 and Ser992) to
the band of hydrophilic residues that line the central
aqueous pore in ABCB1 (Fig. 3). Both of these observations can be rationalized with the molecular models
for ABCB1.
The homology models predict that both V982C and
G984C, located within the centre of the helix, experience little change in molecular environment upon ATP
binding, which is in agreement with the biochemical
data. TM12 is predicted by homology modelling to
rotate by approximately a quarter of a turn following
ATP binding, which is also in agreement with the biochemical data. This rotation is accompanied by a displacement towards Tyr953 (TM11), the nearest
neighbour of Val982 in the closed-state model. Despite
this motion, Val982 does not form a close contact with
Tyr953, and the local environment is therefore
unchanged and does not impact on the accessibility of
the residue to the fluorescent probes. In support of
this, no change in labelling was observed. In addition,
the position of Gly984 does not change between the
closed and open states of the model, and would not
result in a change in the polarity of the environment.
This rigidity is clearly reflected in the labelling experiment, which demonstrated little change in residue
accessibility among the catalytic states.
A hydrophilic band of residues in the TMD lines the
central cavity of ABCB1 (Fig. 3) and presumably contributes to the solvent accessibility of the residues in
this region. M986C and S992C (Fig. 3) on TM12
straddle the boundaries of this hydrophilic band, and
also face directly into the presumed translocation pore.
These two residues were readily labelled by the fluorescent probes, and displayed differences in accessibility
between the conformational states examined. It has

been suggested that conformational transitions may
alter the nature of the residues lining the translocation
pore [10,31], e.g. from hydrophobic to hydrophilic.
This type of switch may be responsible for the cycling
of affinity of ABCB1 for drug substrates during the
translocation process [24]. Such observations have been
made in both the ABCB1 homology models [30] and
the low-resolution crystal structure of ABCB1 [6].

FEBS Journal 277 (2010) 3974–3985 ª 2010 The Authors Journal compilation ª 2010 FEBS


E. Crowley et al.

Role of TM12 in bioenergetic coupling in ABCB1

A

B

C

Fig. 3. Molecular modelling of the TMDs in ABCB1. Representations of the TMDs of ABCB1 obtained from molecular modelling are shown,
with the NBDs removed for clarity. (A) The TMD of ABCB1 predicted to represent the basal (nucleotide-free) conformation. (B) The TMD of
ABCB1 predicted to occur in the nucleotide-bound conformation of the protein. The two TMDs of ABCB1 are shown with helices from
TMD1 (N-terminal) in grey and those from TMD2 (C-terminal) in black. The TMDs display a hydrophilic band of residues (cyan) that lines the
central cavity, and these are shown in the ‘space-fill’ representation. Relative to TM12, the hydrophilic band is located at a depth that corresponds to the region bounded by residues Met986 and Ser992, which are depicted in purple. (C) The TMD helices (cylinders) neighbouring,
or in the vicinity of, TM12 (ribbon). The helices are shown in the nucleotide-free (bold) or bound (pastel) conformations: orange, TM9; gold,
TM10; red, TM11. All other helices have been removed from the diagram to aid clarity. The diagram also demonstrates (comparison of bold
and pastel representations) that TM12 undergoes relatively little motion in switching between these conformations. The structures are

shown in the panel as viewed from the translocation pore; the relative environments of V982C (cyan) and G984C (blue) are unaltered by
nucleotide binding. The nearest neighbouring residue, Tyr953, is shown in red space-fill representation.

Surprisingly, although FM labels G984C, the homology model suggests that this residue faces into the lipid
bilayer. However, G984C is not in a very densely
packed region, and it may be possible for FM to gain
access to the residue via the translocation pore. In
addition, the loss of labelling of G984C with FM following progression to a vanadate-trapped state suggests that labelling is not optimal and therefore is very
sensitive to even minor environmental changes. S992C
and F994C are believed to be located at the boundary
of the membrane. Indeed, Ser992 faces into the translocation pore near the entrance and is highly solvent
exposed. Consequently, both residues are accessible to
labelling by FM. Moreover, Phe994 is located within
the prominent kink in TM12, which was first identified
by the homology model of ABCB1 and subsequently
confirmed in the crystal structure [6,30].
It is conceivable that this kink may facilitate (or
dampen) transmission of movement initiated by events
in the NBDs to conformational changes in TM12. For
example, upon ATP binding, the NBDs will form a
dimer to enable hydrolysis of nucleotide. The resultant
hydrolytic cleavage of ATP will result in disengagement of the dimer because of the considerable repulsion between ADP and Pi. TM12 is directly linked to
NBD2, and is therefore ideally placed to transmit these
conformational changes. The communication would

extend in both directions, and the central region of
TM12 would act as a stationary element about which
the conformational changes occur. Similarly, the binding of substrates is thought to stimulate ATP hydrolysis by facilitating conformational changes associated
with NBD dimer assembly. This might occur through
communication between the drug-binding site(s) and

TM12. In fact, mutations in TM12 were demonstrated
to affect transport or ATPase activity [32,33], in particular, the stimulation of ATP hydrolysis by vinblastine
and nicardipine [15]. These two compounds are known
to interact at pharmacologically distinct (allosterically
linked) sites in ABCB1 [34], and this supports the
notion of TM12 acting as a key conduit. Moreover,
the observation that mutations in TM12 could alter
stable ATP binding by the NBDs further supports the
tight coupling imparted by TM12 on the process of
ATP hydrolysis. Further biochemical and structural
studies will reveal the exact contribution of individual
residues in TM12 to drug binding and the role of
the TM12 anchor region identified here in allosteric
communication.
A previous investigation has also demonstrated that,
upon ATP binding, the extracellular faces of the two
helices can form a zero-length cross-link, indicating a
close approach [35]. This close approach of the helices
is relaxed following progression of ATP hydrolysis.

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Role of TM12 in bioenergetic coupling in ABCB1

E. Crowley et al.

Moreover, there is a large amount of evidence demonstrating that TM6 and TM12 are intimately involved

in numerous aspects of the molecular mechanism of
ABCB1. The present investigation focused on TM12,
and it is clear that the helix does undergo conformational changes, with the centre of the helix being rigid
and motion being amplified at the extracellular and
intracellular ends of the helix.

Experimental procedures

Fluorescent labelling of single-cysteine isoforms
of ABCB1

Materials
Octyl-b-d-glucoside, C219 antibody and Ni2+–nitrilotriacetic acid His Bind Superflow resin were obtained from
Merck Chemicals (Nottingham, UK). Dimethylsulfoxide,
Na2ATP, AMP-PNP, sodium orthovanadate and cholesterol were purchased from Sigma Aldrich (Poole, UK).
Crude Escherichia coli lipid extract was obtained from
Avanti Polar Lipids (Alabaster, USA). Insect-Xpress
medium was purchased from Lonza (Wokingham, UK) and
Excell 405 from SAFC Biosciences (Andover, UK). CM,
FM and BM were purchased from Molecular Probes
(Leiden, The Netherlands).

Site-directed mutagenesis of TM12 in
ABCB1 – introduction of cysteines
Mutants were constructed with QuikChange or Altered
Sites II mutagenesis systems with a pAlter-MCHS or pFastBac1-MCHS template. The MCHS cDNA encodes an
ABCB1 isoform devoid of cysteines with a C-terminal His6
tag and numerous strategically inserted restriction enzyme
sites. Full details of the construction of mutant ABCB1
isoforms have been given in previous publications [25,36].

Expression, purification and reconstitution of
ABCB1
Recombinant baculovirus was generated using the Bac-toBac baculovirus expression system, as previously described
[25,36] and according to the manufacturer’s instructions
(Invitrogen). Trichoplusia ni (High-five) cells were infected
with recombinant baculovirus at a multiplicity of infection
of 5, and harvested 72 h postinfection by centrifugation
(2000 g, 10 min). For comparative analysis of protein
expression, 2 · 106 cells were resuspended in NaCl ⁄ Pi supplemented with 2% (w ⁄ v) SDS, and proteins were resolved
by SDS ⁄ PAGE. ABCB1 was detected with the C219 antibody following immunoblotting [37].
For large-scale expression of ABCB1 isoforms, 1.5 · 109
T. ni (High-five) cells were infected, and cell membranes
were isolated by nitrogen cavitation and density gradient
ultracentrifugation and stored at )80 °C for up to 1 year

3982

[25,36]. ABCB1 isoforms were purified by immobilized
metal affinity chromatography (Ni2+–nitrilotriacetic acid
resin), and reconstituted by the detergent adsorption technique [25,36]. Confirmation of reconstitution was performed by examining the relative migration of lipid and
protein through sucrose density (0–30% w ⁄ v) gradients.
Protein concentration following reconstitution was determined with an adapted Lowry colorimetric assay with BSA
as standard (DC-Brad Protein Assay; BioRad) [38].

The topography of TM12 was assessed by following the
labelling kinetics of each single-cysteine mutant isoform
with three fluorescent thiol-reactive probes. The probes
display distinct physicochemical properties, with variations
in charge, size and hydrophobicity [25]; for example, CM is

hydrophobic, FM is hydrophilic and BM is zwitterionic.
Purified, reconstituted ABCB1 isoforms (2 lg) were incubated with 10 lm CM, BM or FM for 0, 10, 30, 60, 120
and 300 min in the dark at 20 °C. The ligand was added
from concentrated stocks in dimethylsulfoxide, and the final
solvent concentration was maintained at < 0.05% (v ⁄ v).
A 100-fold molar excess of probe to protein was used to
facilitate labelling and prevent significant depletion of the
probes. The reaction was stopped by the addition of
100 lm dithiothreitol, which binds avidly to unreacted
maleimide probe, and subsequently placed on ice. The protein was diluted 1 : 1 with buffer (50 mm Tris ⁄ HCl, pH 7.4,
150 mm NH4Cl, 5 mm MgSO4, 0.02% NaN3) to reduce
glycerol content, and centrifuged for 30 min at 125 000 g
and 4 °C to remove unbound probe. The pellet was washed
and then resuspended in 20 lL Laemmli sample buffer, and
proteins were resolved by 7.5% (v ⁄ v) SDS ⁄ PAGE. Nonspecific association of the fluorescent probe with the protein
and lipid membrane was determined using a cysteine-free
ABCB1 isoform. The G324C mutation, located on a freely
accessible extracellular loop, has previously been demonstrated to be freely accessible to each maleimide probe [28];
labelling of the isoform containing this mutation was therefore assigned the value of 100% after 300 min. Both the
cysteine-less and G324C isoforms were incubated with
10 lm probe for 300 min and treated identically to the
other isoforms. The extent of labelling for each single-cysteine mutant was therefore determined by comparison with
G324C. In order to calculate the specificity of labelling for
each single-cysteine mutant, the background or nonspecific
labelling of the cysteine-less isoform was subtracted. The
propensity for labelling was calculated with the following
equation:
À
Á
Liso À Lcys

Á  100
Lẳ
L324C Lcys

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E. Crowley et al.

Role of TM12 in bioenergetic coupling in ABCB1

where L is extent of labelling (%), Liso is the extent
of isoform labelling, Lcys is labelling of the cysteine-less
isoform, and L324C is labelling of the G324C isoform.
The extent of fluorescence labelling for ABCB1 mutant
isoforms was also determined in the nucleotide-bound
state by trapping with AMP-PNP. The ABCB1 nucleotide-bound conformation was generated by the addition of
AMP-PNP (2 mm), followed by a 20 min incubation at
20 °C. Trapping of ABCB1 in the posthydrolytic state was
achieved by the addition of 300 lm orthovanadate (Vi)
and 2 mm ATP, followed by a 30 min incubation at 37 °C
in order to generate the ADPỈVi transition state intermediate [22,24,25]. Fluorescence labelling was subsequently carried out as detailed in the preceding paragraph.
The extent of labelling was determined by examining the
gel using the BioDocIT Imaging System (UVP), with a UV
light source of wavelength 302 nm and a CCD camera. The
gel was subsequently stained with PageBlue to validate
equivalent protein loading. Densitometric analysis (scion
image) was used to quantify the extent of labelling. The
maximum extent of labelling (Lmax) and half-time of labelling (t1 ⁄ 2) were determined by nonlinear regression of the
exponential reaction curve (graphpad prism 4.0) to plots

of labelling as a function of time:
À
Á
L ¼ Lmax 1 À ekt
where L is the percentage of labelling, Lmax is the maximum extent of labelling (%), k is the observed rate constant for labelling (min)1), and t is time (min). The
labelling rate constant was converted to half-time of labelling according to the following relationship:
t1=2 ¼ Ln2=k

Statistical analysis
All data manipulations and statistical analyses were performed using graphpad prism 4.0. Comparison of datasets
for each isoform was performed with Student’s t-test or
ANOVA (where n > 3), applying Dunnett’s test, where significance was determined by a P-value < 0.05. Values
reported correspond to means ± standard errors of the
mean obtained from at least four independent preparations
of ABCB1.

Homology modelling
A homology model of a nucleotide-free, open-state human
ABCB1 was developed from the open-state mouse P-glycoprotein crystal structure (3G5U.pdb), using the swissmodel
homology modelling server [39], with the aim of producing
an open-state homology model of human ABCB1 that
would complement the previous closed-state Sav1866-based
ABCB1 model [30]. The sequence identity between human
ABCB1 and mouse P-glycoprotein is 86%, giving a very

high degree of confidence to the sequence alignment of the
resulting model. To verify that the residue threading of this
open-state ABCB1 model corresponds to the previously
developed closed-state ABCB1 model [30], the sequence
alignments were cross-referenced to ensure that there was

positional correspondence of the residues in both conformations. The series of single-point mutations to cysteine were
performed at positions 976, 978, 980, 988, 989 and 990 in
the open-state ABCB1 homology model, to give a set of six
single-point mutation open-state ABCB1 models. These
models were developed with the method described in Storm
et al. [28]; they provide an alternative conformation to the
set of closed-state ABCB1 point mutations developed in
Crowley et al. [15], and allow a comparison of the local
environment of each residue in both the open and closed
conformation of ABCB1.

Acknowledgements
E. Crowley was generously supported by a Cancer
Research UK Studentship (C362 ⁄ A5502) awarded to
I. D. Kerr and R. Callaghan. M. L. O’Mara is supported
by a University of Queensland Post-doctoral Fellowship.

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