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© The Authors Journal compilation © 2011 Biochemical Society
Essays Biochem. (2011) 50, 63–83; doi:10.1042/BSE0500063
4
Catalytic and transport cycles
of ABC exporters
Marwan K. Al‑Shawi1
Department of Molecular Physiology and Biological Physics,
University of Virginia, 480 Ray C. Hunt Drive, P.O. Box 800886,
Charlottesville, VA 22908‑0886, U.S.A.
Abstract
ABC (ATP‑binding cassette) transporters are arguably the most important
family of ATP‑driven transporters in biology. Despite considerable effort and
advances in determining the structures and physiology of these transporters,
their fundamental molecular mechanisms remain elusive and highly
controversial. How does ATP hydrolysis by ABC transporters drive their
transport function? Part of the problem in answering this question appears to
be a perceived need to formulate a universal mechanism. Although it has been
generally hoped and assumed that the whole superfamily of ABC transporters
would exhibit similar conserved mechanisms, this is proving not to be the
case. Structural considerations alone suggest that there are three overall types
of coupling mechanisms related to ABC exporters, small ABC importers and
large ABC importers. Biochemical and biophysical characterization leads us to
the conclusion that, even within these three classes, the catalytic and transport
mechanisms are not fully conserved, but continue to evolve. ABC transporters
also exhibit unusual characteristics not observed in other primary transporters,
such as uncoupled basal ATPase activity, that severely complicate mechanistic
studies by established methods. In this chapter, I review these issues as related
to ABC exporters in particular. A consensus view has emerged that ABC
exporters follow alternating‑access switch transport mechanisms. However,
1email
[email protected]
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some biochemical data suggest that alternating catalytic site transport
mechanisms are more appropriate for fully symmetrical ABC exporters.
Heterodimeric and asymmetrical ABC exporters appear to conform to simple
alternating‑access‑type mechanisms.
Introduction
The ABC (ATP‑binding cassette) transporters constitute one of the
largest superfamilies of membrane proteins. They are ubiquitous in all
phyla. Conserved motifs in NBDs (nucleotide‑binding domains) of ABC
transporters constitute the defining elements of this family and represent some
of the most conserved motifs in biology. Consequently, many mechanistic
models based on structural observations have been proposed to account
for ABC transporter functionality. These proteins are found on plasma
membranes and internal organelle membranes. ATP hydrolysis is the energy
source for coupled substrate transport by the ABC transporters, thus substrate
transport can be ‘uphill’ against a concentration gradient. These proteins act
as importers and exporters from cells and organelles. The genetic split between
importers and exporters occurred before the divergence of prokaryotes
and eukaryotes [1]. Substrates or ‘allocrites’ for this family of transporters
encompass all types of molecules that need to be transported across a
membrane against a concentration gradient [2]. Accessory substrate‑binding
proteins are utilized by prokaryotic ABC importers that accumulate substrates
into the cell. There are between 45 and 48 annotated ABC transporter genes
in humans and many are associated with diseases or genetic disorders. To ease
confusion in the field, the genes have been organized into seven subfamilies
in the human genome: subfamily ABCA to subfamily ABCG [3]. This
chapter is devoted to presenting current ideas about the coupling of ATP
hydrolysis to the transport function of these ABC transporter pumps, with a
focus on ABC exporters. Coupling models have been highly contentious and
dependent on the type of experimental data considered and the perspective
while building the model. As will be seen, ABC exporters present some unique
challenges in analysing their coupling mechanisms as they are different from
standard primary transporters in many interesting ways.
Structural organization of ABC transporters and
mechanistic implications
ABC transporters contain TMDs (transmembrane domains) that form
the translocation pathway and two NBDs that constitute the power units
(Figure 1). The general structure, topology and conserved motifs have been
thoroughly reviewed in the previous chapter by Zolnerciks et al. Each NBD
contains two nucleotide‑binding motifs, the Walker A and Walker B motifs.
The Walker A motif is involved in binding of nucleotide phosphates and the
Walker B motif is involved in Mg2+ and water co‑ordination at the catalytic
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Figure 1. Topological arrangements of ABC exporters
Topological models of three ABC exporters illustrating different arrangements of the TMDs and
NBDs. Within the TMDs, the numbered cylinders represent transmembrane α‑helices. Relative
locations of conserved motifs of the NBD are also shown.
sites and contains the catalytic glutamates. The NBDs also contain other
conserved motifs that define the ABC family of proteins. These include the
Q‑loop, involved in Mg2+ co‑ordination and binding to a water molecule
and the ABC signature motif (also referred to as the C motif, consensus
sequence LSGGQXQR) that is also involved in ATP binding. Both are located
between the Walker motifs. In ABC exporters alone, directly preceding the
signature motifs, there are X‑loops (consensus sequence TEVGERG) which
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appear to be involved in interdomain communication and cross‑talk between
NBDs and TMDs. Upstream of the Walker A motif is the A‑loop in which
a conserved aromatic residue interacts with the adenine ring of ATP through
π–π interactions. Downstream of the Walker B motif is the D‑loop involved
in indirect co‑ordination of the γ‑phosphate of ATP through a water molecule
and also the H‑loop involved in hydrogen‑bonding the γ‑phosphate of ATP.
Two NBDs come together to form two NBSs (nucleotide‑binding
sites) with two nucleotides sandwiched in at the interface of the two NBDs
(Figure 2). Together, both NBDs (NBD1 and NBD2) are involved in form‑
ing the two functional NBSs (NBS1 and NBS2). In this ‘nucleotide sandwich’
‘head‑to‑tail’ arrangement, each NBS comprises the D‑loop and ABC signa‑
ture motif of one NBD (e.g. NBD2) and the Walker A and B motifs, H‑loop
and Q‑loop from the other NBD (e.g. NBD1 in this example) to form NBS1.
Thus ATP molecules that occupy the two NBSs (NBS1 or NBS2) can be
simultaneously hydrogen‑bonded by residues from each of the NBDs (NBD1
and NBD2). This type of ‘head‑to‑tail’ ATP‑binding site (NBS) formed at
the interface of the opposite NBD subunits was first predicted by Jones and
George [4] and subsequently verified in isolated nucleotide dimer crystal
structures [5] and later in full transporter structures [6].
Some ABC transporters such as Pgp (P‑glycoprotein; ABCB1) and the
homodimeric ‘half‑transporter’ BCRP (breast cancer‑resistance protein;
ABCG2) contain two consensus NBSs (NBS1 and NBS2), both being capable
of full ATP hydrolytic function. In such transporters, the NBDs are inter‑
changeable [7]. Other transporters, such as the MRP (multidrug‑resistance
protein) family members (ABCC family), contain only one fully functional
Figure 2. Structure of nucleotide‑binding domains
Ribbon diagrams of the two nucleotide‑binding domains of the homodimeric ABC exporter
Sav1866 binding two non‑hydrolysable AMP‑PNP (adenosine 5′‑[β,γ‑imido]triphosphate) mol‑
ecules shown as stick figures (PDB code 2ONJ). Each of the two nucleotides is hydrogen‑bonded
to both NBDs and ‘glues’ them together. Two NBSs are formed in the nucleotide‑binding sand‑
wich conformation. (A) View from inside the membrane. (B) Side view.
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and conserved consensus NBS (NBS1) and one less conserved, degenerate and
less hydrolytically active non‑consensus NBS (NBS2) [8,9]. Consequently, the
mechanism of coupled ATP hydrolysis might not be fully conserved between
all ABC transporter subfamilies.
The minimal functional ABC transporter unit capable of transport con‑
sists of a complex of two NBDs and two TMDs. Given that the two NBSs
are formed at the interface of NBD1 and NBD2, cross‑talk between all four
functional domains is expected and observed [10]. These four domains can be
separately encoded [11] or are encoded as one polypeptide, as in the case of
Pgp (ABCB1) and MRP1 (ABCC1) (Figure 1). Proteins containing all four
domains are called ‘full transporters’. Alternatively, transporters are encoded
as multidomain proteins in various combinations, such as the two identical
homologous halves of the homodimeric half‑transporter BCRP (ABCG2)
(Figure 1). Similar to BCRP, bacterial ABC exporters are half‑transporters
with an NBD linked to a TMD in a single polypeptide form. Several ABC
transporters also have additional domains or subunits that aid in regulation
of transport of their particular substrates, such as accessory substrate‑binding
proteins, regulatory domains, regulatory proteins and extra TMDs (e.g. TMD0
of MRP1 in Figure 1). In prokaryotic importers, the NBDs, TMDs, acces‑
sory regulatory domains and substrate‑binding proteins are encoded as dif‑
ferent polypeptides. For any given transporter, each of the two NBDs and
two TMDs may be identical or similar, generating a two‑fold symmetrical
or two‑fold pseudo‑symmetrical unit [12]. Each TMD in ABC exporters is
usually composed of six α‑helices. However, in prokaryotic ABC import‑
ers, other topologies ranging from five to ten α‑helices per TMD have been
observed [13,14]. Thus the TMDs of importers and exporters cannot be easily
interchanged in models of transport.
It is best to consider the coupling mechanisms of these proteins in three
distinct groups on the basis of their structures [15,16]. (i) ABC export‑
ers, which are found in all kingdoms of life, contain six α‑helices per TMD
(12 transmembrane helices in total in the core unit, see Figure 3). The two
TMDs are not simply joined side by side, but have significant twists and each
has two transmembrane helices ‘domain‑swapped’ into the other domain
[17]. In this group, the α‑helices extend well beyond the membrane into
the cytoplasm as ICLs (intracellular loops). Consequently, the NBDs are
approximately 25 Å (1 Å=0.1 nm) from the membrane. Each half‑transporter
or half‑molecule has two short α‑helices, called coupling helix 1 (between
transmembrane helices 2 and 3 and on ICL1 of the N‑terminal half) and
coupling helix 2 (between transmembrane helices 4 and 5 and on ICL2 of
the N‑terminal half). Thus, in a whole transporter, there are four coupling
helices that provide most of the interactions between the TMDs and NBDs.
Coupling helices 1 make contacts with both NBDs during the formation of
the nucleotide sandwich, but are released from contacts with the opposing
NBD in the apo forms (Figure 3). In contrast, coupling helices 2 are always
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Figure 3. Structural illustration of the alternating‑access mechanism
Ribbon diagrams of structural models of human Pgp in different conformations. (A and B)
Nucleotide‑free apo Pgp based on the crystal structure of mouse Pgp (PDB code 3G5U). (A) and
(B) are rotated 90° relative to each other. The N‑terminal half‑molecule is coloured yellow and
the C‑terminal half‑molecule is coloured green to show domain‑swapped regions. X‑loops are
red, coupling helices 1 are light blue and coupling helices 2 are dark blue as space‑filling models.
The two NBDs are separated in space (bottom) and a V‑shaped cavity is open to the cytoplasm
and to the inner leaflet of the bilayer (A). (C and D) Homology and energy‑minimized model
of Pgp generated using crystal structural co‑ordinates of MsbA (PDB code 3B60) containing
two AMP‑PNP (adenosine 5′‑[β,γ‑imido]triphosphate) molecules shown as space‑filling models
(orientations of C and D correspond to orientations of A and B respectively). The AMP‑PNP
molecules bind the two NBDs together in the nucleotide‑binding sandwich conformation. In (D),
a V shaped cavity is open to the extracellular medium in the alternate‑access conformation. (E)
α‑Carbon traces of ten calculated energy‑minimized intermediate structures (rainbow‑coloured)
between the two conformations (A) (red) and (C) (blue) illustrating the large motions associated
with ‘alternating access with a twist’.
domain‑swapped into the other half‑molecule’s NBD. This is not seen in
other ABC proteins. Furthermore, NBD X‑loops interact with the opposite
TMD, mainly through ICL1 [18], but also through ICL2 [19]. Consequently,
switching of conformations and orientations of the TMDs coupled to
ATPase activity at the NBDs/NBSs is expected to be completely different
for ABC exporters and importers. (ii) Group 2 are ‘small’ prokaryotic ‘type I
ABC importers’ that have five or six transmembrane helices per TMD (usu‑
ally 12 in total). The core consists of two TMDs each containing five trans‑
membrane helices that are positioned side by side. An additional N‑terminal
transmembrane helix is occasionally found that is domain swapped into
the other TMD. In contrast with ABC exporters, coupling helices 2 are not
domain‑swapped into the other half molecule, but are associated with their
own NBD. (iii) Group 3 are ‘large’ prokaryotic ‘type II ABC importers’
that have ten transmembrane helices per TMD (20 in total). Here there is no
domain‑swapping between TMDs or NBDs. For further details, see the pre‑
vious chapter by Zolnerciks et al.
All of these widely divergent architectures and distant lineages sug‑
gest that the mechanism of coupled ATP hydrolysis might not be fully
conserved between all ABC transporters. Excellent reviews are available
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describing the mechanisms of ABC importers which are generally thought
to obey ‘alternating‑access’‑type schemes [15,16,20]. Many of the con‑
troversial issues discussed in this chapter regarding basal ATPase, power
strokes, stoichiometries of transport and hydrolysis, symmetry and asym‑
metry have their counterparts in ABC importers, but are beyond the scope
of this chapter.
Mechanistic requirements for coupling of substrate
transport to ATP hydrolysis
In active transport, the uphill change in chemical potential of the transported
allocrite, or work performed, is coupled to and driven by a spontaneous
process that releases energy. ATP hydrolysis powers all primary ABC
transporters in performing their varied pump functions. A pump needs at least
two gates that must be alternately open, but never be open simultaneously
[21]. Here a gate is defined as a protein conformation that stops movement
of the allocrite across it in the closed conformation, but allows movement
in the open conformation. Each of the two gates must open only to one side
of the membrane and not to the same side as the other gate. When both gates
are closed, there is an ‘occluded state’, and the transported entity is trapped
within the protein. A change in chemical potential (from low to high) of the
transported allocrite occurs during this process of occlusion/deocclusion.
The step of the reaction cycle in which this change in chemical potential
of the transported entity occurs is the ‘power stroke’. Chemical energy is
transformed to vectorial energy (osmotic or electrochemical energy) in this
step [22].
In principle, any two obligatorily coupled reactions cycles, such as
chemical and vectorial transport cycles sharing common intermediates, can
drive each other [23]. In ABC proteins, the chemical reaction cycle con‑
stitutes the intermediates and steps of ATP hydrolysis on the transporter.
The vectorial transport cycle represents the sequential protein conforma‑
tions and allocrite‑bound states and steps that are involved in transporting
the allocrite from one side of the membrane to the other side. However,
in active transport, there is an additional requirement that any reaction steps
that would lead to uncoupled partial reactions must be avoided [24]. This is
achieved by interleaving the partial reactions of the chemical reaction and
the vectorial transport cycle such that that they are inextricably interwoven.
Neither the vectorial reaction nor the chemical reaction can occur unless the
other reaction occurs. Coupling and energy conversion thus require that
the partial reactions of ATP hydrolysis control the coordinated opening
and closing of gates and the occlusion of the transported entity. ABC pro‑
teins are very dynamic by nature and exhibit large conformational changes
at most, if not all, of the steps of the hydrolytic reaction and transport
cycles (Figure 3E). Thus, to define the molecular mechanism of transport
and coupling, it is necessary to know the exact step (partial reaction) at
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which the change in chemical potential of the transported entity occurs.
This is a difficult endeavour, which has led to the proposal of many conten‑
tious alternative and sometimes mutually exclusive models of the molecular
mechanisms for the coupling of transport to ATP hydrolysis by the differ‑
ent ABC transporters.
Transport and coupling mechanism of ABC exporters
To consider the mechanism of allocrite transport by ABC exporters, it is
instructive to use the MRP Pgp (ABCB1) to establish ground rules from
which exceptions observed in other ABC exporters can be interpreted
and understood. Pgp is most useful in this role because it was the first
ABC exporter recognized and, as a consequence, and due to its medical
importance, it is the most thoroughly analysed ABC transporter
physiologically, genetically, functionally, structurally, biochemically and
biophysically [25,26]. It is a single polypeptide which appears to have
arisen by tandem duplication of an ancestral half‑transporter and now
contains all four core functional domains with no complicating accessory
subunits [27] (Figure 1). Consequently, it is nearly symmetrical in the two
NBDs and NBSs, which are functionally equivalent and interchangeable [7].
Both NBSs are fully conserved consensus NBSs that are both catalytically
and equivalently active [28,29]. Pgp is pseudo‑symmetrical in the two
TMDs (Figure 3). It can be purified to homogeneity and reconstituted
with full functionality into proteoliposomes of defined composition [30].
Transport substrates of Pgp are well defined and characterized. Most
importantly, changes in drug chemical potential on transport have been
directly recorded in well‑defined proteoliposomes containing only Pgp [31].
A moderate‑resolution X‑ray crystal structure of apo (nucleotide‑free) Pgp
is also available [32].
There has been a tremendous amount of information generated on the
molecular features that allow Pgp to transport drugs or allocrites coupled to
the hydrolysis of ATP. The various approaches used in these studies include
mutational analyses and detailed biochemical, biophysical and pharmaco‑
logical characterizations that have been reviewed thoroughly previously
[25,26,33–35]. It has been shown conclusively that allocrites interact with
Pgp directly. Drug transport by Pgp is thermodynamically coupled to ATP
hydrolysis. Binding of ATP or drugs to Pgp leads to conformational changes.
Pgp pumps drugs directly rather than altering drug distribution indirectly. The
minimal functional unit of Pgp is a monomer. Drugs are extracted from the
cytoplasmic leaflet of the plasma membrane and pumped to the external medi‑
um. Many purine NTPs can power the reaction cycle, implying a large degree
of flexibility in the NBSs. Both catalytic sites (NBSs) were shown to be essen‑
tial for turnover and inactivation of either inhibited turnover. But, at any given
point in the reaction cycle, only one NBS is catalytically active and so catalysis
alternates between the two sites (see below).
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The conundrum of reaction pathway control: ABC
transporters are both tightly coupled and fully uncoupled
under different conditions
Allocrite transport by ABC transporters is tightly coupled to ATP hydrolysis,
but ABC transporters also exhibit the very unusual phenomenon of basal
ATPase activity in the absence of transport allocrites. At first glance, this
phenomenon seems to violate the rule of coupling that states that reactions
that lead to uncoupling must be avoided. With the rules of coupling relaxed,
a ‘reverse water wheel’ (water pump)‑type mechanism was initially proposed
in which Pgp is continually hydrolysing ATP and alternating transport‑site
access (inside and outside) whether the transport‑site is occupied or not [36].
However, this type of model was unsatisfactory in explaining the very tight
coupling of allocrite transport to ATPase activity by Pgp under optimal
conditions as described below. To maintain the rules of coupling, others had
ascribed the basal ATPase activity to an ‘apparent uncoupled ATPase activity’
associated with the transport of an elusive undiscovered endogenous allocrite
(reviewed in [25,26]).
The ATPase activity of Pgp shows three transport allocrite‑dependent
phases: basal (no allocrite), allocrite‑activated (at moderate allocrite concen‑
trations), and allocrite‑inhibited (at high allocrite concentrations) [30,37]. At
optimal concentrations of activating drug (allocrite), the maximal transport
rate (and equivalent coupled ATPase activity) is a characteristic of the particu‑
lar drug and is inversely proportional to the number of hydrogen bonds the
drug can form with Pgp [38]. This indicates direct and tight control of the
ATPase and transport activities of Pgp by the bound drug. Further thermody‑
namic analysis also revealed that, when drug concentrations were optimal, all
ATPase activity proceeded through a very tightly coupled pathway. But, in the
absence of any transport substrate, all ATPase activity proceeds through a dif‑
ferent catalytic pathway [39] (Figure 4). At intermediate drug concentrations,
the ATPase activity partitions between the two different catalytic pathways
in proportion to the activating drug concentration bound to this proportion
of the Pgp population of molecules. Thus there is an allocrite‑dependent
transport and coupled ATPase pathway and a different non‑productive basal
ATPase side reaction. Each of these two pathways has different chemical inter‑
mediates and partial reactions. The model in Figure 4, by having two separate
distinct ATP hydrolytic pathways, reconciles how Pgp can be both tightly
coupled when optimal concentrations of transport allocrites are available and
also completely uncoupled when no transport allocrites are present.
The apparent affinity of Pgp for ATP increases when drugs bind to the
‘inward‑facing’ conformation (Figures 3A and 3B) which facilitates the bind‑
ing of a second ATP molecule (Figure 4, unshaded cycle). Consequently, ATP
hydrolysis and drug transport proceed through the coupled catalytic pathway.
If no drug binding occurs, the apparent affinity for the second ATP remains
low and a second ATP molecule rarely binds to initiate the basal ATPase
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Figure 4. Catalytic and transport cycles of Pgp
Partitioning model of Pgp catalytic cycles. Upper cycle: drug‑activated fully‑coupled activity.
Shaded lower cycle: fully‑uncoupled basal activity. If there is insufficient transport drug to bind
to Pgp, but two ATP molecules end up binding, Pgp partitions to the shaded uncoupled cycle and
hydrolyses ATP without any transport work after passing through a basal transition state (cyan
star). However, if transport drug is present, Pgp partitions to the coupled activity cycle (alternat‑
ing catalytic site transport cycle). Transport drug binds first at the cytoplasmic side (green) which
facilitates the binding of the second ATP molecule at the alternative nucleotide‑binding site. This
leads to the occlusion of ATP and drug to form the ternary complex. After passing through a dif‑
ferent high‑energy transition state (red star), drug is released to the other side of the membrane
(orange). The two transition states (cyan and red stars) represent two different experimentally
demonstrated protein conformations.
pathway and so the level of basal ATPase activity is usually kept low (Figure
4, shaded cycle). Membrane lipid composition and particularly cholesterol
modulates the relative partitioning level between the two pathways [39] by
modulating the ATP‑binding affinity. This may be related to the need to fill
up the drug‑binding cavity with cholesterol for efficient coupling between
the ATPase reaction cycle and the drug‑transport cycle [40] as described in
Chapter 3 by Zolnerciks et al.
Another ABC MRP, PDR5 (pleiotropic drug resistance 5), found in yeast,
has one canonical NBS and one degenerate NBS. In contrast with Pgp, PDR5
appears to proceed mostly through the uncoupled pathway by a ‘reverse water
wheel’‑type mechanism [41].
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Thus it now appears that a very important feature of ABC exporters is to
control the reaction pathway, through a large set of alternative dynamic inter‑
mediates. This allows for a much greater flexibility in transport and in selec‑
tion of different substrates. As a consequence, the mechanistic and transport
pathways followed by different ABC transporters are likely to be different and
to be optimized for each one’s particular function.
Alternating‑access switch models represent the current
consensus view of the mechanism of ABC exporters
Switch‑based models were formulated for Pgp by analogy to the putative
mechanisms of ABC importers and from detailed structural analyses of full
ABC transporters and isolated NBDs as described in the previous chapter
by Zolnerciks et al. The alternating‑access switch models are derived from
the Jardetzky conformational model for transport [42]. All such models have
three consistent elements [12]. (i) Binding of transport substrate to the TMDs
of the apo form in the ‘high‑affinity inward‑facing orientation’ initiates the
transport cycle by allowing the reaction cycle to proceed. (ii) ATP binding
induces the formation of the closed nucleotide sandwich structure. ATP acts
as molecular glue that holds the two NBDs together. The binding energy
gained by the NBDs/NBSs is transmitted to the TMDs which then change
their access to the ‘low‑affinity outward‑facing orientation’ on the other
side of the membrane. Thus the gate to the inside is closed and the gate to the
outside of the membrane is opened, and the affinity of the transported entity
for the ABC transporter changes (switches) from high affinity (low chemical
potential of substrate) to low affinity (high chemical potential). Consequently
the ATP‑binding step can be considered as the power stroke in which the
chemical potential of the transported entity changed. (iii) ATP hydrolysis leads
to the formation of extra negative charge, thus opening the closed nucleotide
sandwich structure by charge repulsion [43]. Charge repulsion driving a
conformational change is a common attribute of most, if not all, mechanisms
envisaged. The opening of the nucleotide sandwich structure facilitates Pi
release and ADP dissociation, which in turn allows the TMDs and access‑gates
to reset to the high‑affinity orientation on the original side of the membrane.
The first premise of switch‑type models is the establishment of stable
dimeric nucleotide sandwich structures containing two ATP molecules sym‑
metrically positioned in the two NBSs. However, these structures have never
been observed in the presence of physiologically relevant ATP concentrations
and have only been observed when the NBDs were made non‑functional by
mutation of a critical residue, by removing the essential Mg2+ cofactor or by
use of non‑hydrolysable ATP analogues [26]. Under physiological conditions,
the ATP concentration is so high that it is unlikely that Pgp is ever found in
a nucleotide‑depleted apo form [44] as required by some models. EPR spec‑
troscopy of the intact ABC exporter MsbA (a homodimeric half‑transporter
homologous with Pgp) shows that ATP binding in the absence of Mg2+ does
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not close the NBDs and Mg2+ itself was without effect [45]. Another premise
of switch‑type models is that the affinity for transported drug should decrease
on ATP binding before initiation of the catalytic transition state. This was
indeed demonstrated in some experiments employing non‑hydrolysable ana‑
logues of ATP with Pgp [46] or BCRP (ABCG2) [47]. However, in other
experiments using physiological nucleotides, the affinities of drugs for Pgp
measured in the resting state, before ATP binding, was essentially the same
as their affinities during the catalytic transition state [48,49], complicating
the application of this premise to Pgp. As discussed in the previous chapter,
the evidence for switch type models from interpretations of static structural
snapshots may be overwhelming, but these mechanisms do not satisfactorily
explain all available biochemical and biophysical experimental observations for
Pgp under physiological‑like conditions.
Alternating catalytic site mechanisms are appropriate for
fully symmetrical ABC exporters
Alternating catalytic site models, first proposed for Pgp [50], have several
consistent elements. (i) ATPase activity coupled to drug transport alternates
sequentially between the two kinetically equivalent NBSs, thus generating
asymmetry at the NBSs. (ii) Each ATP hydrolytic event leads to generation
of a distinct charge‑repulsion event and hence a distinct power stroke
capable of driving an allocrite across the membrane (Figure 5). (iii) The
allocrite‑transport cycle and the ATP‑hydrolysis cycle are inextricably
interleaved as required by coupling considerations. (iv) Such models have
a theoretical transport stoichiometry of one ATP hydrolysed per allocrite
moved that has been demonstrated experimentally for Pgp under optimal
conditions [51]. In sharp contrast, a value of two ATP hydrolysed per
substrate moved was determined in some tightly coupled ABC importers [52].
Again this would imply that different coupling mechanisms exist for ABC
exporters and ABC importers.
A prediction of alternating catalysis was that one ATP should be occluded
at a particular catalytic site (either NBS1 or NBS2) before being committed to
hydrolysis in that NBS. The occlusion represents an interleaved partial step
of the ATPase reaction with a limited transfer of ATP’s chemical potential to
Pgp which is probably employed to close the inward‑facing drug‑access gate.
This predicted single asymmetrical occluded ATP at either NBS was directly
demonstrated in a catalytically inactivated Pgp mutant where the Walker B
catalytic glutamate residues of the NBDs were changed to alanine [53]. The
mutations eliminated two of the critical negative charges (Figure 5) required
for initiation of the power stroke by charge repulsion on ATP hydrolysis.
Later, the single asymmetrical occluded ATP was also observed in normal
Pgp by the use of the non‑hydrolysable ATP analogue adenosine 5′‑[γ‑thio]triphosphate [54,55]. Binding of a second ATP triggers transient occlusion
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Figure 5. Model of Pgp’s alternating catalytic site power stroke
Left: ATP binding to NBS1 leads to the occlusion of the ATP at NBS2 so that it is committed to
hydrolysis and attacked by a water molecule. Right: ATP hydrolysis at NBS2 generates a surplus
of negative charge after the reaction proton diffuses away. Electrostatic repulsion then propels
the two NBDs apart at NBS2 while closing NBS1 further. These power stroke movements are
transmitted to the TMDs via the coupling helices. After product dissociation and ATP binding at
NBS2, the next power stroke is generated by ATP hydrolysis at NBS1. Further power strokes
are generated at the two sites in alternating fashion.
of the low‑affinity ATP bound in the previous cycle, followed by immediate
hydrolysis of this occluded ATP. Thus, in fully intact Pgp, one can either trap
one catalytic transition state or one occluded ATP [53,55], but never trap two
ATPs at the same time as required by switch‑type models. The occluded ATP
leads to asymmetry in the NBDs, NBSs and TMDs [56] and this asymmetry
constitutes the conformational memory required by the alternating cata‑
lytic mechanism [50]. Such alternating catalysis (Figure 5) is also supported by
molecular dynamics computations [57,58] and by electron microscopy struc‑
tural studies [59].
Variations on the themes and hybrid mechanisms
Sauna and Ambudkar [60] have proposed a hybrid ‘occluded
nucleotide‑binding switch’ model for Pgp. Here ATP binds to the NBDs
and induces the closed nucleotide sandwich structure as above, but with
no associated substrate affinity changes. Then the switch of high affinity to
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Essays in Biochemistry volume 50 2011
low affinity by the drug‑binding site is produced by occlusion of one ATP
molecule (tight binding and going from high to low chemical potential) that
occurs randomly at a given NBS. The occluded nucleotide is committed to
hydrolysis and is hydrolysed. Next, dissociation of ADP or exchange for
ATP resets the drug‑binding site to a high‑affinity form. This model does not
overcome problems of intrinsic reaction pathway uncoupling generated by
NBS asymmetry and TMD symmetry as discussed below.
Other hybrid mechanisms include the ‘processive clamp model’ [61].
ATPase activity in the homodimer of fragmented NBDs of Mdl1 (ABCB10),
was not co‑operative in both NBDs and therefore hydrolysis must be sequential
between the NBDs (processive). It was concluded that ATP binding in separat‑
ed NBDs leads to dimerization of the NBDs (clamp) to form the NBSs and that
this clamp step provides the power stroke for the reorientation of the TMDs
from inward‑facing to outward‑facing conformation as in the switch models.
Then a random stochastic ATP hydrolysis at either NBS occurs, followed by
the hydrolysis of the other ATP at the other NBS, which leads to separation
of the NBDs and relaxation of the TMDs to the inward‑facing conforma‑
tion. The processive clamp model was based on results obtained using isolated
NBDs (fragmented protein) and on extrapolation from structural observations
obtained with prokaryotic ABC importers, which appear to function differ‑
ently from ABC exporters. This hybrid processive clamp model is more similar
to the switch models. On the other hand, the recently proposed hybrid ‘affin‑
ity‑switch model’ [62] first proposed for the single consensus NBS‑containing
ABC Cl− channel CFTR (cystic fibrosis transmembrane conductance regulator;
ABCC7) and extended to Pgp (ABCB1) is more similar to alternating catalytic
site models. Here it was concluded that ATP binding to the consensus NBS2
(NBS1 is always ATP‑occupied) leads to a closed dimer formation that drives a
change of orientation of the TMDs from inward‑facing to outward‑facing, but
without an affinity change for the transported substrate. Hydrolysis of ATP
at the catalytically competent NBS2 is the power stroke that brings about the
affinity change of the substrate by further subtle conformational changes in
the TMDs. Opening of NBS2 follows, and dissociation of Pi and ADP allow the
transporter and TMDs to reset to the initial inward‑facing conformation.
Symmetry and asymmetry considerations
Early on it was recognized that the original formulation of the alternating
catalytic site mechanism [50] had a problem in postulating one symmetrical
allocrite‑binding site with two gates, controlling access to the inside and
outside, located between the symmetrical TMDs. Essentially, an alternating
asymmetrical power stroke cannot drive a symmetrical alternating‑access
model without violating the principles of coupling and thus leading to intrinsic
uncoupling. In other words, the two sets of coupled reactions (chemical ATP
hydrolysis and vectorial allocrite transport) have to be either fully symmetrical
(as in the case of switch‑type models) or fully asymmetrical to obtain coupling.
© The Authors Journal compilation © 2011 Biochemical Society
M.K. Al‑Shawi
77
Otherwise, uncoupling chemical and transport reaction shortcuts are feasible
(bypass pathways) that are not strictly interleaved with the other coupled
reaction pathway.
The alternating catalytic site (two‑cylinder engine) transport model [63]
was proposed to account for the requirements of asymmetry throughout the
reaction/transport cycle of the homodimeric LmrA. (i) In this model there
are two allocrite channels that alternate in transport and each allocrite chan‑
nel is located on a given half‑transporter (or homologous half of Pgp) or at the
interface between the two transmembrane helices. (ii) It is implied that there
are two gates in each drug channel (four gates in total) controlling the orien‑
tation of the drug‑binding site within. Whereas the two‑cylinder model gives
an excellent fit to biochemical observations, it was abandoned due to presumed
incompatibilities with the available structural data. In particular there was
a lack of structurally observed independent channels with the added pres‑
ence of domain swapping between the two half‑transporters. However, given
the dynamic nature of LmrA, these structural considerations might, in fact, be
more of a misperception than true incompatibilities.
The original alternating catalytic site model for Pgp has been modified
to include asymmetries of the NBSs and TMDs to avoid uncoupling and to
be more consistent with recent structural studies. Figure 6 represents such a
Figure 6. Alternating catalytic site and asymmetrical Y transport model of Pgp
Circles, squares and hexagons represent different conformations of the N‑terminal and
C‑terminal catalytic sites (NBS1 and NBS2). Both NBSs bind ATP with similar affinities and both
hydrolyse ATP, but not in the same catalytic cycle. The two NBSs interact strongly and can‑
not hydrolyse ATP independently. Clockwise from top left: the transport and catalytic cycle is
initiated on binding of a drug molecule (blue circle) to TMD2 through an open gate to the inner
leaflet of the plasma membrane. ATP binding to NBS1 closes the gate, occludes the drug, and
occludes and commits to hydrolysis the ATP bound previously at NBS2. Hydrolysis of ATP pro‑
ceeds at NBS2 generating a high chemical potential form of the enzyme (catalytic transition state,
hexagon). Relaxation of the high chemical potential protein conformation in NBS2 performs
the transport work by changing the conformation of the asymmetrical TMDs such that TMD2
exposes the drug to the shared permeation pathway to the extracellular medium. ADP release
from NBS2 resets the transporter to the alternative conformation. An inner leaflet gate is now
opened on TMD1 and the shared permeation pathway gate to the outside is closed. Binding of
the next drug molecule (red circle) to TMD1 allows the cycle to continue in the alternative con‑
formation with ATP hydrolysis at NBS1. At any given point in the cycle, there is no symmetry in
either the NBSs or TMDs.
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Essays in Biochemistry volume 50 2011
model based on results obtained in our laboratory. This scheme, which we
call the ‘alternating catalytic site and asymmetrical Y drug transport model’,
has a minimum of three gates, two occluded drug states and two occluded
nucleotide forms. It shares many elements with the alternating catalytic site
(two‑cylinder engine) transport model [63].
One may speculate that an ancestral, four‑gate and two‑channel,
homodimeric ABC transporter was an original form of ABC exporters, but
as the need for transport of large substrates emerged, the mechanism evolved.
The alternating catalytic site and asymmetrical Y transport model has two
asymmetrical gates controlling transported substrate entry in accordance with
the asymmetry required by the two NBDs and two NBSs. Structural elements
of the exit gate are shared, but in an asymmetrical form in the two half‑cycles
(Figure 6). In other words, the two NBSs and TMDs are always asymmetrical,
as has been experimentally demonstrated. Transport of the drug through a
given channel is facilitated by the observed asymmetrical transmembrane helix
movements as described in the ‘solvation‑exchange mechanism’ of drug trans‑
port by Pgp where a large change in affinity for the transported drug is not
essential [38].
Nature’s re‑simplification of ABC exporters by inhibiting
ATP hydrolysis at one nucleotide‑binding site
The alternating catalytic site and asymmetrical Y transport model describes
a complicated scheme devised by Nature to allow for the transport of
many large substrates by a transporter that is simultaneously symmetrical
in structural elements, but completely asymmetrical in action. An easy
simplification of the ‘asymmetrical Y transport channel’ would be to
remove one of the arms of the Y such that the transporter is asymmetrical
in all actions at all times. There is evidence that Nature has performed
this simplification many times during the evolution of asymmetrical ABC
exporters. Examples are found in the heterodimeric TAP (transporter
associated with antigen processing) 1/TAP2 (ABCB2/ABCB3) [8],
or the asymmetrical MRP family (ABCC family) (Figure 1) [64] or even
the asymmetrical Cl− channel CFTR (ABCC7) [62]. This simplification is
achieved by changing one of the consensus NBSs to a non‑consensus NBS
that cannot or only poorly hydrolyses ATP. As was detailed for the TAP1/
TAP2 system, ATP hydrolysis by the consensus NBS alone still allows
peptide transport. This was demonstrated directly by studies that further
mutated the degenerate NBS to prevent any possible ATP hydrolysis at this
degenerate site (reviewed in [65]). As a direct consequence of asymmetry in
the NBSs, one of the asymmetrical gates controlling entry of substrate into the
transporter (one arm of the Y) is always closed due to the continual presence
of ATP in the non‑consensus NBS that cannot be hydrolysed to open this
gate. In such an asymmetrical system, transport more or less conforms to the
allosteric alternating‑access model [42].
© The Authors Journal compilation © 2011 Biochemical Society
M.K. Al‑Shawi
79
Conclusion
It has generally been presumed that all ABC transporters share similar
types of transport and coupling mechanisms even though there is no
compelling theoretical justification for this presumption. In the ABC
transporter family, there are distant genetic lineages; very diverse and not
fully conserved structural and topological elements; very diverse structural
transmission and coupling pathways; different symmetries and asymmetries
of nucleotide‑binding domains and TMDs; different nucleotide‑binding sites
that are fully functional, partly functional or completely non‑functional;
differing levels of coupled and uncoupled ATPase activity; and differing
coupling stoichiometries. All of this diversity argues against a single
universal transport and coupling mechanism. Rather, the mechanistic
and transport pathways followed by different ABC transporter classes
and individual transporters are likely to be optimized for their particular
function. Most structural studies, and especially crystallographic ones, are
performed outside the realm of dynamics and time domains and cannot
account for chemical potential changes or protein dynamics. On the other
hand, kinetic analyses and some biochemical and biophysical studies
can be performed under physiologically relevant conditions and within
dynamic time domains. As ABC proteins are very dynamic, they do not
like to be ‘coerced’ into ordered crystals. Furthermore, the detergents
used do remove membrane lipids that might be required to maintain
physiologically relevant structures. Thus many structural analyses are
performed in extra‑physiological domains where proteins do not operate.
To move forward, more complementary and interlaced kinetic, biochemical,
biophysical and structural studies are urgently needed. Additionally, a new
thrust towards elucidating and understanding the nature and pathways of
protein dynamics during the transport and catalytic cycles is needed to better
define the transport mechanism of ABC exporters in molecular terms. Such
a molecular‑based understanding is an absolute prerequisite for any rational
medical intervention in the myriad diseases mediated by the functioning or
the lack of functioning of ABC exporters.
Summary
•
•
•
ABC transporters are structurally heterogeneous and are likely to have
three overall types of coupling mechanisms specific to ABC exporters,
small ABC importers and large ABC importers.
Even within these three ABC transporter classes, the catalytic and
transport mechanisms are not fully conserved.
ABC transporters perform dynamic reaction pathway control.
Partitioning between highly coupled transport cycles and uncoupled
basal ATPase is controlled to aid in selection of diverse transport
substrates.
© The Authors Journal compilation © 2011 Biochemical Society
80
•
•
•
Essays in Biochemistry volume 50 2011
Alternating‑access switch transport models of ABC exporters are in
good agreement with structural studies, but not in total agreement
with biochemical studies on full and homodimeric ABC exporters.
Alternating catalytic site transport models of ABC exporters are in
general agreement with biochemical studies, symmetry and asymmetry
considerations, and can accommodate many current structural observ‑
ations, but are not in full agreement with all structural observations.
Heterodimeric ABC exporters and full ABC exporters containing a
non‑functional nucleotide‑binding site probably evolved to conform to
simple alternating‑access transport‑type mechanisms.
This work was supported by the National Institutes of Health [grant number
GM52502].
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