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ABC transporters: the power to change

Douglas C. Rees, Eric Johnson and Oded Lewinson
Abstract | ATP-binding cassette (ABC) transporters constitute a ubiquitous superfamily of
integral membrane proteins that are responsible for the ATP-powered translocation of
many substrates across membranes. The highly conserved ABC domains of ABC transporters
provide the nucleotide-dependent engine that drives transport. By contrast, the
transmembrane domains that create the translocation pathway are more variable. Recent
structural advances with prokaryotic ABC transporters have provided a qualitative molecular
framework for deciphering the transport cycle. An important goal is to develop quantitative
models that detail the kinetic and molecular mechanisms by which ABC transporters couple
the binding and hydrolysis of ATP to substrate translocation.

ABC
(ATP-binding cassette). A set
of conserved sequence motifs
that defines an eponymous
family of ATPases. ABC is often
used interchangeably with
nucleotide-binding domain
(NBD).
Division of Chemistry and
Chemical Engineering 114‑96,
Howard Hughes Medical
Institute, California Institute
of Technology, Pasadena,
California 91125, USA.
Correspondence to D.C.R.
e‑mail: dcrees@caltech.edu
doi:10.1038/nrm2646
Any object that enters or leaves a cell, whether a nutri-
ent, a virus or a waste product, must penetrate one or
more enclosing membranes. The magnitude of this
phenomenon might be estimated, for example, by the
need of an actively growing Escherichia coli cell to take
up ~106 glucose molecules per second1,2 to support its
requisite metabolic demands. Cells must not only be able
to import preferred substrates, but they also often have
the capability to use a wide range of alternative nutrients
when available. With a few exceptions (such as oxygen
and nitrogen), the movement of small molecules, ions and
even some macromolecules across membranes is medi-
ated by specialized membrane proteins that are known as
transporters. To accommodate the diversity of molecules
that a cell might need to acquire from the environment,
many different transporters are encoded in the genomes
of organisms. In E. coli, for example, ~10% of the genome
has been classified as participating in transport processes3
and, overall, more than 550 different types of transporters
have been identified4. The importance of transport activity
can be appreciated from the non-trivial metabolic costs
of pumping molecules across cell membranes, which are
estimated to consume ~10–60% of the ATP requirements
of bacteria5,6 and humans7, depending on conditions.
One of the largest classes of transporters is the ATP-
binding cassette (ABC) transporter superfamily 8–10. These
transporters use the binding and hydrolysis of ATP to
power the translocation of a diverse assortment of sub-
strates across membranes, ranging from ions to macro-
molecules. ABC transporters function as either importers,
which bring nutrients and other molecules into cells, or
as exporters, which pump toxins, drugs and lipids across
membranes (BOX 1). Members of the ABC transporter
family are present in organisms from all kingdoms of
life; whereas exporters are found in both eukaryotes and
prokaryotes, importers seem to be present exclusively
in prokaryotic organisms. ABC transporters constitute the
largest protein family in E. coli, including ~80 distinct sys-
tems that represent 5% of the genome11, whereas ~50 ABC
transporters are present in humans12. Members of the seven
families of human ABC transporters13 participate in choles-
terol and lipid transport, multidrug resistance, antigen
presentation, mitochondrial Fe homeostasis and the ATP-
dependent regulation of ion channels (including the cystic
fibrosis transmembrane conductance regulator and the
sulphonyl urea receptors). Mutations in these proteins
have been associated with a range of disorders, including
cystic fibrosis, hypercholesterolaemia and diabetes.
ABC transporters have a characteristic architecture
that consists minimally of four domains (FIG. 1a): two
transmembrane domains (TMDs) that are embedded
in the membrane bilayer, and two ABCs (or nucleotide-
binding domains (NBDs)) that are located in the cytoplasm.
At the sequence level, the superfamily of ABC transporters
is identified by a characteristic set of highly conserved
motifs that are present in the ABCs. By contrast, the
sequences and architectures of the TMDs are variable,
reflecting the chemical diversity of the translocated sub-
strates. Beyond these four domains, additional elements can
fuse to the TMDs and/or ABCs of ABC transporters and
probably have regulatory functions14. For prokaryotic ABC
transporters that function as importers, substrate trans-
location is also dependent on another protein component,
a high-affinity binding protein that specifically associates
with the ligand in the periplasm for delivery to the appro-
priate ABC transporter 15 (FIG. 1a). Originally recognized
by Heppel16, these binding proteins are released follow-
ing osmotic shock. Therefore, the associated transport
systems, which are now recognized as ABC transporters,
were initially identified as ‘shock-sensitive’.

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Box 1 | The ins and outs of ABC transporters
Cellular survival requires the generation and maintenance of electrical and chemical
concentration gradients across the generally impermeable cell membrane.
ATP-binding cassette (ABC) transporters are key participants in this process, and
typically use the favourable chemical energy of ATP hydrolysis to translocate molecules
across membranes in a thermodynamically unfavourable direction. A given ABC
transporter might function as either an importer or an exporter, moving molecules in or
out of cells, respectively, but no example is known of an ABC transporter that can
function physiologically in both directions. ABC transporters that function as importers
are found in prokaryotes, in which they mediate the uptake of nutrients, such as amino
acids, sugars and essential metals. Substrates of ABC importers vary greatly in size and
chemical nature, ranging from oligopeptides and oligosaccharides to small ions. ABC
exporters are found in both prokaryotes and eukaryotes. In humans, ABC exporters are
crucial participants in the export of lipids, fatty acids and cholesterol, and their
malfunction underlies various diseases. Perhaps the most studied ABC transporter is
human P-glycoprotein (PGP), which maintains cholesterol distribution across the
leaflets of the plasma membrane75,76. Unfortunately, PGP also extrudes other lipophilic
compounds, including chemotherapeutic agents, resulting in the multidrug resistance
of tumour cells.
Whether functioning as importers or exporters, ABC transporters probably share a
high degree of mechanistic similarities. For both exporters and importers, an
‘alternating access’ model for transport has been suggested17,31,48. The key feature of
this model is the presence of a substrate-binding site that can alternately access either
the extracellular or the intracellular side of the membrane, corresponding to the
outward-facing and inward-facing conformations of the transporter, respectively.
The binding and hydrolysis of ATP drive the conformational changes that result in the
alternating exposure of this binding site to the two sides of the membrane. The relative
binding affinities of the two conformations for substrate will largely determine the net
direction of transport. In particular, the outward-facing conformation of an importer is
expected to have a higher affinity for substrate than the inward-facing conformation,
whereas the opposite will be true for exporters.
Following the determination of the crystal struc-
ture of the E. coli vitamin B12 importer BtuCD in 2002
(ReF. 17), structural studies of intact ABC transporters
have exploded over the past 2 years18–26 (TABLe 1). We
refer to recent reviews that highlight these structural
advances27–33. A brief discussion of developments in the
crystallography of ABC transporters can be found in
Supplementary information S1 (box). Here, we build on
this structural foundation and focus on the comparative
and mechanistic aspects of ABC transporters, and empha-
size developments that involve prokaryotic members
BtuCD of this superfamily.
The vitamin B12 uptake
system that is composed of
Structural organization
the integral membrane protein
subunit BtuC and the The overall architecture of ABC transporters can be
ATP-binding cassette subunit illustrated by two structures that were determined by the
BtuD. group of Kaspar locher: the BtuCD importer in complex
with its associated binding protein BtuF21 (FIG. 1b), and
Sav1866
A bacterial ATP-binding
the multidrug exporter Sav1866 from Staphylococcus
cassette exporter from aureus 18 (FIG. 1c). The four domains of the BtuCD trans-
Staphylococcus aureus that is porter consist of four individual polypeptide chains,
homologous to eukaryotic including two copies of the BtuC (TMD) subunits and
multidrug resistance
two copies of the BtuD (ABC) subunits. By contrast, each
transporters.
subunit of Sav1866 contains a TMD and an ABC fused
MalFGK2 together, such that the complete transporter is a dimer. In
A maltose transporter, which is both cases, the transporter exhibits a molecular two-fold
composed of two homologous axis that superimposes the two TMDs, as well as the two
integral membrane subunits —
MalF and MalG — and two
ABCs. The straddling of the TMDs around the two-fold
copies of the ATP-binding axis creates the translocation pathway across the mem-
cassette subunit MalK. brane, whereas the two ABCs are orientated such that
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the conserved sequence motifs (FIG. 2a) are positioned
at the dimer interface (FIG. 2b,c). Distinct quaternary
structure arrangements have been observed that corres-
pond to outward- or inward-facing conformational
states (BOX 1; see below).
ABC subunits. ABC subunits are dominated by the
NBD, which can be further divided into two constituent
domains: a catalytic core domain, which contains the
conserved P-loop or Walker A motif (GXXGXGK(S/T)),
a Walker B motif (ϕϕϕϕD, of which ϕ is a hydrophobic
residue), a Q-loop and an H-motif (or switch region);
and a more structurally diverse α-helical domain 34,
which contains the ABC signature motif lSGGQ
(FIGS 2a,b). The relative orientation of the helical and
catalytic domains is sensitive to the nucleotide state35.
In an intact transporter, the ABC subunits pack together
in a head-to-tail fashion, such that the P-loop of one
subunit is orientated towards the signature motif of the
other (FIG. 2c). An important consequence of this head-
to-tail orientation is that the nucleotide-binding site is
positioned at the subunit–subunit interface, in a manner
that was originally proposed from modelling studies36
and was crystallographically characterized for the non-
transporting ABC protein rad50 (ReF. 37) (see below).
This arrangement was subsequently observed with iso-
lated transporter ABC subunits38 and in the intact ABC
transporters Sav1866 (ReF. 18) and the maltose trans-
porter MalFGK2 (ReF. 23). It is generally observed that the
ATP-bound state is associated with the most extensive
interface between ABC domains, whereas the structures
of nucleotide-free transporters exhibit conformations
with greater separations between the ABC domains.
TM domains. Although it is often commented that the
TMDs of ‘typical’ ABC transporters contain a total of
12 TM helices (6 per domain), the membrane-spanning
subunits are structurally heterogeneous and three dis-
tinct sets of folds are currently recognized. These folds
are designated33 type I ABC importer, type II ABC
importer and ABC exporter folds (FIG. 3a–c). Type I and
type II importer nomenclature reflects the historical
sequence in which members of the relevant families were
characterized in sufficient detail, such that their distinct
TMD folds could be recognized.
The type I ABC importer fold was originally observed
in the ModB TM subunit of the molybdate transporter 22,
and was subsequently found in the MalF and MalG TM
subunits of MalFGK2 (ReF. 23) and the Met transporter
MetI26. This fold is organized around a minimal set of
5 TM helices in each subunit, as observed in MetI (FIG. 3a).
ModB, MalF and MalG contain additional helices, totalling
6, 8 and 6 TM helices, respectively. Consequently, for the
complete transporters, a total of 10, 12 and 14 TM helices
are found in the Met, molybdate and maltose transporters,
respectively. using the helix numbering of MetI, helices
TM2–5 adopt an ‘up–down’ topology that lines the per-
meation pathway, whereas TM1 wraps around the outer,
membrane-facing surface and contacts the other 4 TM
helices. The additional helices in ModB, MalF and MalG
primarily interact with the other subunit.
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a Outward facing Inward facing polarities through the membrane. Helices TM5 and
TM10 dominate the interface between TMDs.
The ABC exporter fold, which was originally estab-
Binding
protein lished for the Sav1866 exporter 18 and subsequently in
Periplasm the homologous MsbA24, contains 6 TM helices per
subunit, or 12 for the complete transporter (FIG. 3c). In
TMD TMD TMD TMD the outward-facing conformation that is adopted by
Sav1866, the TM-spanning region is organized into two
‘wings’ that are composed of helices TM1 and TM2 from
Substrate Cytoplasm
one subunit and TM3–6 of the other. This occurs in an
ATP ADP
ABC ABC ABC ABC unanticipated, domain-swapped arrangement. A nota-
ble aspect of this arrangement is that helices TM1–3 are
related to TM4–6 by an approximate two-fold rotation
b c around an axis in the membrane plane. Furthermore,
Binding protein exporters contain long intracellular loops (ICls) that
extend the TM helices ~25 Å beyond the membrane
surface and into the cytoplasm.
Although the detailed folds of the TMDs can vary, they
Periplasm all interact with the helical domains of the ABC domain
through coupling helices that are located in the loops
TMD TMD between TM helices17. For importers, the coupling helices
contain the consensus eAA motif 39,40, which was pre-
viously recognized to contribute to the interface between
Cytoplasm the TMD and the ABC domain. Although the arrange-
ments are distinct, structurally equivalent coupling helices
are present in the loops between TM3 and TM4, and TM6
ABC ICL
and TM7, of the type I and type II ABC importer folds,
respectively. The equivalent region in exporters involves
ICl2, which is found between helices TM3 and TM4 and
ABC contacts the ABC domain of the other subunit. In all struc-
tures, the region of the ABC domain that interacts with
the TMD primarily involves the Q-loop in the α-helical
domain. The Q-loop is conformationally variable,
with the conserved Gln participating in nucleotide bind-
Figure 1 | Molecular architecture of aBc transporters. a | A cartoon of the modular
ing. Changes in the Q-loop region are proposed to be
organization of ATP-binding cassette (ABC) transporters, involved in the coupling of nucleotide hydrolysis to the
Naturewhich are| composed
Reviews of two
Molecular Cell Biology
transmembrane domains (TMDs) and two ABC domains (or nucleotide-binding conformational states of the TMD41.
domains). The binding protein component that is required by importers is also shown.
Two conformational states of the ABC transporter — outward facing and inward facing, Additional TMD folds? Are there additional classes
with the substrate-binding site orientated towards the periplasmic (extracellular) and of TMD folds in ABC transporters that have yet to be
cytoplasmic (intracellular) regions, respectively — are depicted to show the alternating identified? The answer to this is almost certainly yes,
access mechanism of transport (BOX 1). b | The Escherichia coli vitamin B12 importer and one indication for this answer comes from a phylo-
BtuCDF22 (Protein Data Bank (PDB) code 2QI9). The core transporter consists of four genetic classification based on the protein sequences of
subunits: the two TMD BtuC subunits (purple and red) and the two ABC BtuD subunits
ABC domains that delineate three main classes of ABC
(green and blue). This complex also contains one copy of BtuF, the periplasmic binding
protein (cyan). c | The Staphylococcus aureus Sav1866 multidrug exporter18 (PDB code
transporters29,42. Two of these classes contain membrane-
2ONJ). Sav1866 consists of two subunits (green and dark blue), which contain a fused spanning domains that have either fused ABCs and
TMD and ABC domain. The nucleotides that are bound in this structure are shown by TMDs (sequence class 1) or have the TMDs and ABCs
yellow space-filling models. Molecular figures in this article were prepared with on independent polypeptide chains (sequence class 3).
MolScript and Raster3D77,78 using coordinates from the PDB79. ICL, intracellular loop. A comparison between this sequence-derived classifica-
tion and the structurally characterized families of ABC
transporters reveals some interesting features that are
The type II ABC importer fold, which is found in relevant to the existence of structurally uncharacterized
BtuC17 and in Hi1471, a homologous transporter from TMD folds.
Haemophilus influenzae20, contains 10 TM helices per Proteins in sequence class 1 of ABC transporters
subunit, or a total of 20 for the complete transporter typically function as exporters, and because the sequence
(FIG. 3b). These helices pack together in an intricate similarities are high, it is likely that the membrane
topology that positions helix TM2 through the centre topologies of all of these proteins corres pond to
of the subunit in proximity to most of the other helices. the structure of the ABC exporter fold in Sav1866
The amino- and carboxy-terminal halves of the BtuC (ReF. 18) . By contrast, sequence divergence between
subunit exhibit similar helix packing, particularly the binding-protein-dependent importers that are
for helices TM2–5 and TM7–10, but with opposite found in sequence class 3 is substantially higher.

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Table 1 | Structurally characterized ABC transporter systems, as classified by the TMD fold*
Transporter organism Nucleotide state orientation PDB code resolution references
Type I ABC importer fold
ModABC Archaeoglobus fulgidus Apo Inward 2ONK 3.1 Å 22
MalFGK2 Escherichia coli ATP-bound Outward 2R6G 2.8 Å 23
ModAB Methanosarcina acetivorans Apo Inward 3D31 3.0 Å 25
MetNI Escherichia coli Apo Inward 3DHW 3.7 Å 26
Type II ABC importer fold
BtuCD Escherichia coli Apo or (VO2)4 Outward 1L7V 3.2 Å 17
Hi1470 and Haemophilus influenzae Apo Inward 2NQ2 2.4 Å 20
Hi1471
BtuCDF Escherichia coli Apo Occluded 2QI9 2.6 Å 21
ABC exporter fold
Sav1866 Staphylococcus aureus ADP-bound Outward 2HYD 3.0 Å 18
AMPPNP-bound Outward 2ONJ 3.4 Å 19
MsbA Salmonella typhimurium AMPPNP-bound Outward 3B60 3.7 Å 24
ADP–vanadate-bound Outward 3B5Z 4.2 Å 24
Escherichia coli Apo Inward 3B5W 5.3 Å 24
Vibrio cholerae Apo Inward 3B5X 5.5 Å 24
*The nomenclature of the fold classification is taken from ReF. 33. Note that while the outward-facing conformation generally corresponds to the ATP (or suitable
analogue)-bound state, exceptions are evident in the structures of ADP bound to Sav1866 and nucleotide-free BtuCD. ABC, ATP-binding cassette; Btu, vitamin B12
importer; Mal, maltose transporter; Met, Met transporter; Mod, molybdate transporter; PDB, Protein Data Bank; TMD, transmembrane domain; (VO2)4,
cyclotetravanadate.

On the basis of the sequence comparisons of the ABC
domains, 12 families of importers have been identi-
fied29,42. Structurally characterized transporters with
the type I ABC importer fold (the maltose, molybdate
and Met transporters), correspond to the sequence-
derived oligosaccharides and polyols (OSP), mineral
and organic (MOI) and d-l-Met and derivatives (DlM)
families of transporters (nomenclature taken from
ReF. 29), respectively, and are present in one branch of
the sequence-derived phylogenetic tree. By contrast,
transporters with the structurally characterized type II
ABC importer fold correspond to the sequence-derived
Fe-siderophores, vitamin B12 and hemin (ISVH) family,
which occupies a separate branch of the sequence-
derived phylogenetic tree to proteins that have the
type I ABC importer old.
Intriguingly, a third branch of the sequence-derived
phylogenetic tree exists that is distinct from these other
two branches and contains the hydrophobic amino acid
(HAA; such as the leu, Ile, Val (lIV) system) and mono-
saccharide (MOS; such as ribose (rBS)) families of
ABC transporters. As the HAA and MOS families are
clearly separated from the branches that contain trans-
porters with either type I or type II importer folds, they
are potential candidates for a distinct TMD fold. One
member of the MOS family, the rbsC protein, probably
contains 10 TM helices, of which no obvious mapping
to the 10 TM helices of BtuC was reported43 (it should
be noted, however, that an eAA sequence is present
in the loop between putative helices TM6 and TM7 of
rbsC, which corresponds to the location of this motif
in BtuC17).
In addition to the sequence comparisons of the ABC
domains, the structures of the binding proteins also
suggest that the HAA and MOS branch could exhibit
a distinct TMD fold. The structures of the binding pro-
teins for ABC transporters vary in their topological
threading between domains44–46, and can be classified
into three distinct categories (designated I, II and III,
which confusingly overlaps with the nomenclature used
for the TMD folds). A tantalizing correlation exists in
transport systems that have a type I ABC importer fold
for the TMD and a binding protein type I fold (for exam-
ple, ModA, Male and MetQ), whereas transport systems
with a type II ABC importer fold have a binding protein
type III fold (for example, BtuF). Transport systems that
exhibit the binding protein type II fold (for example,
rbsB, livJ and livK) correspond to the HAA and MOS
sequence families of ABC domains, which might suggest
that they also exhibit a distinct structural architecture
for the TMDs. The validity of this speculation will be
established by the crystal structure determination of an
intact transporter from one of these families.
Kinetic mechanism of ABC transporters
The similarities in ABC structures support a common
mechanism by which ABC transporters, both importers
and exporters, orchestrate a series of nucleotide- and
substrate-dependent conformational changes that result
in substrate translocation across the membrane27. The
‘alternating access’ model47,48, in which the substrate-
binding site alternates between outward- and inward-
facing conformations, provides a productive framework
for this mechanistic analysis (BOX 1). The successful

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a P-loop or Walker A ABC signature H-motif state of the transporter is achieved, at which point ATP
...GXXGXGK(S/T)... ...LSGGQ... ...H... hydrolysis will drive substrate translocation.
N C
...Q... ...φφφφD...
Q-loop Walker B ATP hydrolysis. An important mechanistic question for
ABC transporters concerns the ratio of ATP hydrolysed
b c per translocated substrate, and whether or not there is
complete coupling of these processes under physiological
P P
conditions. Although the mechanistically relevant ratio
H H for ABC transporters is probably 2 ATP molecules per
B B
substrate transported49, this ratio has only been reliably
Q Q observed in an in vitro system for the tightly coupled
OpuA transporter 50. More generally, ATPase and trans-
ABC ABC
port rates differ by 1–3 orders of magnitude49,51–53, and
completely uncoupled ATPase activity can be observed in
the absence of substrate49. Whereas two ABC domains are
Figure 2 | Structure and dimer interactions of an aBc subunit. a | A linear always required for activity (see below), only one func-
representation of the protein sequence of an ATP-binding cassette| Molecular
Nature Reviews (ABC) subunit,
Cell Biology
tional ATPase site can support transport activity in certain
showing the relative positions along the polypeptide chain of the conserved amino-acid
motifs. b | Stereoview of the ABC subunit BtuD17 (Protein Data Bank (PDB) code 1L7V).
systems, including the His permease54, although this is
The P-loop (P), Walker B motif (B), Q-loop (Q), H-motif (H) and ABC signature motif (ABC) not a universal property of ABC transporters29.
are indicated along one surface of the subunit. A cyclotetravanadate bound to the P-loop The enzymatic hydrolysis of ATP requires the presence
is shown as a ball-and-stick model. c | The nucleotide-mediated ABC dimer of the of two sets of properly positioned groups: for binding the
Staphylococcus aureus Sav1866 multidrug exporter18 (PDB code 2ONJ). The two ABC phosphates and for catalysing the attack of water on
domains are shown as ribbons (green and dark blue), whereas the sandwiched AMPPNP the γ-phosphate. For a large family of nucleotide-binding
(an ATP analogue) nucleotides are yellow space-filling models. The P-loops and the proteins, including ABC transporters, the conserved
signature motifs are depicted in red and cyan space-filling models, respectively. The Walker A and Walker B motifs55 participate by binding
coupling helices of the TMDs in contact with the ABCs are shown as purple ribbons. The the nucleotide phosphates and the Mg 2+ that is coordin-
view is down the molecular two-fold axis.
ated to the nucleotide, respectively. These interactions are
shown for Sav1866 in FIG. 4a. The Mg 2+ interaction
involves the Asp residue in the Walker B motif (Asp502
operation of transporters that move molecules across in Sav1866). In addition to these binding interactions, two
membranes against a concentration gradient requires sets of residues are commonly found in the active site to
the elimination of short-circuiting. This is achieved by catalyse the rate of ATP hydrolysis: one group serves as a
preventing the uncoupled processes from occurring in general base that promotes the attacking water, whereas
their energetically favourable directions — that is, leak- the other electrostatically stabilizes phosphate oxygens56,57.
age of the accumulated substrate across the membrane Candidate residues for the general base include Glu503,
or the futile cycling of ATP hydrolysis. which is adjacent to the Walker B motif, Gln422 from
These general considerations can be shown through an the Q-loop and His534 in the H-motif region, which all
F1-ATPase idealized kinetic model for transport (see Supplementary cluster in the vicinity of the cleaved phosphate (FIG. 4a).
A stable, water-soluble information S2 (box)). In this model, it is assumed that The residue that serves as the primary catalytic residue
subassembly of the ATP and ADP are bound by the outward- and inward- is ambiguous. Although there is strong evidence in some
membrane-bound ATP
synthase, of which the active
facing conformations of the transporter, respectively, and systems that the Glu adjacent to the Walker B motif is the
site for ATP synthesis and that ATP hydrolysis drives the conversion from outward- crucial catalytic residue38,58,59, this might not be universally
hydrolysis is located at the to inward-facing states. The net rate of substrate trans- true, as an important role for the H-motif His has been
interface between homologous location is the difference between the rates of import and observed in other systems60.
α- and β-subunits.
export. To function as a pump that imports substrates An interesting comparison can be made with other
Nitrogenase across the membrane, the rate of the forward reaction, ATPases, including the F1-ATPase61 (FIG. 4b) and the nitro-
The enzyme system that or influx into the cell, needs to be maximized, whereas genase Fe-protein62 (FIG. 4c), which are central participants
catalyses the ATP-dependent the backward reaction, or efflux, needs to be minimized. in ATP synthesis and biological nitrogen fixation, respec-
reduction of dinitrogen to For importers, the optimization of the substrate uptake tively. The F1-ATPase shares structural similarity to the
ammonia during the process of
biological nitrogen fixation. The
rate against a concentration gradient, while minimizing fold of the catalytic domain of the ABC transporters,
nitrogenase Fe-protein ATP hydrolysis, can be kinetically achieved through a whereas the nitrogenase Fe-protein is in a distinct,
contains the binding site for combination of effects: a higher affinity for substrate in although related, branch of nucleotide-binding domains
ATP. the outward-facing conformation than in the inward- that includes G proteins and ras p21 (ReFS 63,64). The
facing conformation; the stimulation of ATPase activity interaction of Mg 2+ ATP with the Walker A and B motifs
G protein
One of a family of GTP-binding on substrate binding to the outward-facing conforma- is similar among ABC transporters, F1-ATPase and
proteins that are involved in tion of the transporter; and a higher rate of nucleotide nitrogenase Fe-protein, although the conformation of
signal transduction, in which exchange of ATP for ADP in the unliganded state of the the ATP varies between these structures. The catalytic
GTP binding and hydrolysis transporter, which resets the transporter. For exporters, residue Gluβ188 of the F1-ATPase is spatially analo-
and subsequent nucleotide
exchange drive a series of
the opposite set of relationships is required. The key to gous to the Q-loop Gln residue, whereas there are no
conformational changes that minimizing the futile cycling of nucleotide is to keep the counterparts to Glu503 or His534 in this protein. Gly128
are used in signalling cascades. rate of ATP hydrolysis minimal until the proper liganded of nitrogenase Fe-protein, which has been implicated

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a Type I importer b Type II importer c Exporter
5 10
2 4 5 5
3 6
8 3
9 1
1 the transporter cycles through the stages of ATP bind-
3 1
7 4
4 2
6
Figure 3 | Polypeptide folds of a single transmembrane domain in aBc transporters.
The type I ABC importer fold of the Met transporter MetI (Protein Data Bank (PDB) code
Nature Reviews | Molecular Cell Biology
3DHW; part a), the type II ABC importer fold of BtuC (PDB code 1L7V; part b) and the
ABC exporter fold of the Staphylococcus aureus Sav1866 multidrug exporter (PDB code
2ONJ; part c). The top panels are traces of the polypeptide fold for each structure, with a
colour gradient that ranges from red at the amino terminus through yellow and green to
blue at the carboxyl terminus. The molecular two-fold axis, which corresponds to the
normal to the membrane plane, is vertical. The bottom panels are ribbon representations
of the transmembrane (TM) helices in these structures, viewed from the periplasmic
surface rotated 90° from the top view. The molecular two-fold axis is depicted in red in
the lower figures. All of the TM helices (TM1–5) are shown for MetI; the BtuC helices are
divided into two groups, TM1–5 and TM6–10; and the Sav1866 helices are divided into
groups TM1–3 and TM4–6. In each set, the helices are coloured red to blue proceeding
along the polypeptide chain from the N terminus to the C terminus to show the internal
symmetry that is present in BtuC and Sav1866. These internal repeats are related by a
rotation axis that is orientated approximately vertically in the plane of the figure.
in the stabilization of the γ-phosphate, superimposes
with the H-motif His534, whereas Asp39 in the switch I
region superimposes with Glu503. A counterpart to the
residue that is thought to promote the attack of water
in nitrogenase Fe-protein (Asp129 of the other subunit
in the nitrogenase Fe-protein dimer) is not present in
Sav1866. Thus, in these other characterized ATPases, no
consistent pattern of catalytic relevance can be assigned
to potential residues in the ABC transporter, which sug-
gests that there is no unique solution to the organiza-
tion of the active-site residues in Walker A- and Walker
B-containing enzymes.
The conformation of ABC transporters that is cata-
lytically competent for nucleotide hydrolysis involves
AAA+ ATPase the binding of an ATP molecule at the interface between
(ATPases associated with two ABC domains, with the terminal phosphates of the
diverse cellular activities). nucleotide bound between the P-loop on one ABC and
A large family of structurally the lSGGQ signature motif of the other (as shown for
conserved ATPases in which
assembly of the ATPase active
Sav1866 with AMPPNP, an ATP analogue, in FIGS 2b, 4a).
site requires subunit Consequently, ATP binding and hydrolysis involve the
oligomerization. participation of both ABC domains. Individual subunits
NATure reVIeWS | Molecular cell Biology
© 2009 Macmillan Publishers Limited. All rights reserved
REVIEWS
cannot productively bind and hydrolyse ATP, which
explains why two ABC domains are required. Again,
comparisons of the active-site arrangement for ATP
hydrolysis in other ATPases are informative. The resi-
dues that correspond to the lSGGQ signature motif in
the active sites of F1-ATPase and nitrogenase Fe-protein
ATPase (the positively charged residues Argα373
and lys10′, respectively) are provided by neighbouring
subunits (FIG. 4a–c). This bipartite organization of the
active site, in which essential catalytic groups are posi-
tioned on different subunits, is found in such diverse sys-
tems as G proteins65, AAA+ ATPases64 and helicases66, and
has the important implication that the rate of nucleotide
hydrolysis can be controlled by the process of assembling
the active site67,68.
Nucleotide-dependent changes in transporter NBDs.
The key to the functioning of ABC transporters is the
2 positional relationships between the two ABC domains,
which depend on the nucleotide state (see above). As
ing, hydrolysis and product dissociation, the interface
between the two ABC domains switches from a closed
interface, with the bound ATP sandwiched between the
conserved sequence motifs of different domains, to a
more open interface that is characteristic of the non-ATP
states. These nucleotide-dependent conformational trans-
itions drive the conformational changes in the TMDs,
resulting in substrate translocation. The magnitude of the
variability in the relative positions of ABC domains can
be shown through a comparison of the ABC domains
from different transporters (FIG. 5).
Some considerations that are relevant to the super-
position and comparison of structures are discussed in
Supplementary information S3 (box). It should also be
emphasized that the assumption is made in this type of
comparison that the observed distribution of structures
reflects conformations that are mechanistically relevant
to the transport cycle, rather than the idiosyncrasies of
the different transporters. Although common themes
are observed, one must keep this caveat in mind when
considering specific details. Following the superposition
of one ABC domain from each of a set of different trans-
porters, substantial variation is evident in the positioning
of the second ABC domain. A measure of the separation
between ABC domains can be provided by the distance
between the P-loop and lSGGQ signature motifs on dif-
ferent subunits (defined here as the distance between the
positions of Cα atoms corresponding to BtuD residues
Gly38 and Gly129, respectively). The intersubunit separa-
tions between the two conserved motifs range from ~11 Å
in the ATP-bound form of Sav1866 to 14 Å, 16 Å and
~28 Å in the nucleotide-free forms of BtuD, Hi1470
and MetN, respectively 26. A more detailed analysis indi-
cates that the relationships between the non-superimposed
subunits in the different structures can be approximately
described in terms of two basic operations: first, a
hinge- or tweezer-type rotation that juxtaposes the ABC
domains by closing up the interface69; and second, a twist-
type rotation that corresponds to a sliding or translational
shift of ABC domains along the interface20.
VOluMe 10 | MArCH 2009 | 223
REVIEWS
a Sav1866 b F1-ATPase c Nitrogenase Fe-protein
Argα373 Lys10′
Asp502 Aspβ256
Gln422 Asp125
LSGGQ Asp39
Glu503 Gluβ188 Asp129′
His534
Gly128
Figure 4 | Nucleotide–protein interactions in the active sites of various aTPases.
AMPPNP (an ATP analogue) bound to the ATP-binding cassette
Nature Reviews(ABC) transporter
| Molecular Cell Biology
Staphylococcus aureus Sav1866 multidrug exporter19 (Protein Data Bank (PDB) code
2ONJ; part a), the ADP–AlF4 site of the F1-ATPase61 (PDB code 1H8E; part b) and the
ADP–AlF4 state of the nitrogenase Fe-protein80 (PDB code 1M34; part c). The nucleotide
is depicted with yellow bonds, whereas Mg2+ (or equivalent) is cyan and AlF4 (when
present) has purple atoms. The P-loop is indicated by the green trace connecting the
Cα atoms of adjacent residues at the back of each structure, whereas the Walker B motif
Asp residue from the same subunit is shown with red bonds on the left. In Sav1866, the
potential catalytic residues are Glu503 (adjacent to the Walker B motif), Gln422 in the
Q-loop and His534 of the H-motif. The backbone atoms of the LSGGQ signature motif in
the dimer-related ABC subunit are depicted with blue bonds. For the F1-ATPase, the
catalytic residue Gluβ188 occupies the same spatial location as the Q-loop, whereas
Argα373 from the adjacent subunit coincides with the signature motif. For nitrogenase
Fe-protein, Asp39 in the switch I region and Gly128 correspond to the Q-loop and
H-motif region, respectively, and the catalytic residue Asp129′ from the adjacent
subunit has no obvious counterpart in the ABC transporters. Lys10′ from the
adjacent subunit is positioned similarly to the signature motif in the ABC transporters.
Interconversion of alternating conformations. The
interconversion between the closed ABC interface in
the ATP state and the more open interfaces in non-ATP
states drives the switching of the translocation path-
way between the outward- (ATP-bound) and inward-
facing (ADP-bound or nucleotide-free) conformations,
respectively 52. It should be emphasized, however, that
this statement is a generalization that might not always
be completely valid. For example, the structures of
nucleotide-free BtuCD 17 and ADP-bound Sav1866
(ReF. 18) adopt the outward-facing conformations,
although they were both crystallized in the absence of
ATP. Furthermore, electron spin resonance spectro-
scopy studies that involve site-directed spin labelling
indicate that ATP alone is insufficient to drive the
closure of the maltose transporter NBDs — the pres-
ence of the maltose-binding protein is also required70.
By contrast, the conformation of the outward-facing
gate of BtuCD depends only on the docking of the bind-
ing protein and is insensitive to binding of AMPPNP,
whereas the inward-facing gate probably opens under
these conditions71. This final study suggests that there
might be mechanistic distinctions between different
families of ABC transporters and highlights the need
for caution when extrapolating results from one system
to another.
The conformation of the permeation pathway of
the TMD is connected to the nucleotide state of the
transporter through interactions between the helical
domains and the coupling helices of the ABC domains
and TMDs, respectively (FIG. 2c). For the three sets of
224 | MArCH 2009 | VOluMe 10
© 2009 Macmillan Publishers Limited. All rights reserved
TMD folds in transporters that have been structurally
defined (TABLe 1), an ensemble of structural states are
available that can be used to identify the nature of these
conformational transitions in the TMDs, at least in
broad outline (see below).
For type I ABC importers, structures are available
that represent a progression of the conformations of
the translocation pathway, ranging from the occluded
or outward facing (for example, the maltose trans-
porter) to inward facing (for example, the molybdate
transporter) to wide-open, inward facing (for example,
the Met transporter). Superimposing the structurally
conserved helices TM2–4 (helix numbering of MetI)
onto the MalF subunit of the maltose transporter, the
transformations that are required to superimpose
the appropriate helices of MalG with the corresponding
regions of ModB and MetI correspond to rigid body
rotations of ~20° and 32°, respectively 26. This is equiva-
lent to a hinge-type motion, in which the rotation axis
is approximately perpendicular to the symmetry axis of
the transporter and passes near a conserved Pro resi-
due (Pro67 in MetI) at a kink in helix TM2 near the
periplasmic surface.
Structures that represent outward-facing (for
example, BtuCD), occluded (for example, BtuCDF)
and inward-facing (for example, Hi1470 and Hi1471)
states of homologous type II ABC importers are also
available and exhibit differences in their tertiary and
quaternary arrangements of subunits20,22. At the terti-
ary structure level, the main difference between the
outward and occluded structures are the local rear-
rangements in the helices TM3–5 of one subunit
that are not part of the rigid core. Of these, the most
important difference is a 20° shift in the helix axis of
TM5, because this helix is part of the dimer interface
between BtuC subunits. The conversion to the inward-
facing conformation involves a twist of ~9° about an
axis that is normal to the molecular two-fold axis. As
a result of the repositioning of helices TM3–5 and the
overall twist, the translocation pathway shifts from
outward to inward facing.
For ABC exporters, the structures of Sav1866 and
MsbA in outward-facing conformations are available.
These correspond to the ATP-bound state. Two lower-
resolution structures of MsbA homologues have also
been solved that adopt inward-facing conformations
that are described as closed inward and open inward.
In the open inward conformation, the NBDs are widely
separated. A comparison of the MsbA structures indi-
cates that the transition from the open to closed inward
conformation corresponds to a ~30° hinge axis rota-
tion. The formation of the tight ABC interface involves
a distinct twist of TM4 and TM5 that displaces the ABC
domains along the subunit–subunit interface until the
closed ATP state is achieved. During these rearrange-
ments, the packing of the TM helices changes, such
that whereas TM1–3 and TM6 from one subunit and
TM4 and TM5 from the other subunit are together in
the inward state, in the outward state, TM1 and TM2
from one subunit and TM3–6 from the other subunit
are juxtaposed.
www.nature.com/reviews/molcellbio

MetNI
A Met transporter, which is
composed of the integral
membrane protein MetI and
the ATP-binding cassette
subunit MetN.
NATure reVIeWS | Molecular cell Biology
ABC motif
subunit 1
Subunit 2 Subunit 1
28 Å
14 Å 11 Å
16 Å
MetN BtuD Sav1866
Hi1470 MalK P-loop
subunit 1
Open Closed
(ATP)
Figure 5 | relationships between dimeric aBc
Nature Reviews
structures. The polypeptide | Molecular
fold of BtuD Cell
(Protein Biology
Data
Bank (PDB) code 1L7V) is shown with the two subunits
traced in different shades of blue. The positions of the
P-loop and ATP-binding cassette (ABC) signature motifs
(defined by the positions of the Cα atoms of Gly38 and
Gly129, respectively) in BtuD are indicated. The positions
of the P-loop and ABC signature motifs, following
superposition onto the right-most BtuD (subunit 1) of one
ABC domain of the Staphylococcus aureus Sav1866
multidrug exporter (red; PDB code 2ONJ), MalK (yellow;
PDB code 1Q12), Hi1470 (cyan; PDB code 2NQ2) and the
Met transporter MetN (green; PDB code 3DHW), are
designated by the coloured spheres. AMPPNPs (an ATP
analogue) that are bound at the ABC dimer interface of
Sav1866 (PDB code 2ONJ) are shown with purple bonds.
The closed interface that is characteristic of the ATP-bound
state, as observed in Sav1866 and MalK, is associated with
an intersubunit separation between the P-loop and ABC
signature motifs of ~11 Å. In nucleotide-free structures, the
separation increases from 14 Å in BtuD to 16 Å in Hi1470 to
~28 Å in MetN. The large separation between ABC subunits
in MetN probably underlies the phenomenon of
trans-inhibition, in which Met binding to a regulatory
domain of MetI stabilizes an ATPase-inactive form by
sterically preventing the formation of the catalytically
essential closed interface between these domains.
Regulation of ABC transporter activity
The activity of ABC transporters might be regulated at the
level of protein function through the action of domains
fused to the NBDs and/or TMDs14. It should also be con-
sidered that non-covalently associating subunits could
also be involved. Trans-inhibition is one example of such
a regulatory process, in which the uptake of an external
ligand is inhibited by intracellular concentrations of the
same ligand, as observed by Kadner for the uptake of
Met 72. recent crystallographic analyses of the ModBC25
and MetNI26 systems have established a structural basis for
this effect as involving ligand binding to the C-terminal
domain of the ABC subunits. Although the domains in the
two systems are distinct, they each participate in the bind-
ing of allosteric ligands to other proteins, and therefore
© 2009 Macmillan Publishers Limited. All rights reserved
REVIEWS
seem to have been recruited by ABC transporters for
these properties. In each case, the consequences of ligand
binding to the regulatory domain are similar in that the
liganded C-terminal domains sterically separate the ABCs
and prevent their association. Because the ABC subunits
must associate to form the ATP-bound state, keeping
them separated will reduce the rate of ATP binding and
thereby inhibit the overall transport cycle.
Intriguingly, a large family of soluble ABC proteins
that contain two NBDs has been found to participate
in antibiotic resistance by certain pathogens, and, of
particular note, the C terminus of the S. aureus VgaA
protein has been implicated in regulating the response
to antibiotics73. This observation suggests that a similar
mechanism to that which underlies the phenomenon of
trans-inhibition by ABC transporters might be relevant
to this system.
Future directions
Our current understanding of ABC transporters can be
compared to that of a play, in which we know the names
of essentially all of the actors, what some of them do and
what some of them look like, and we also have a syn-
opsis of the plot. However, we are missing the detailed
script (or scripts, if there is more than one basic plot) that
describes what all of the actors are doing, and how and
why they are doing it. reconstruction of this script will
require progress in many directions.
Given the prevalence of ABC transporters through-
out life, surprisingly few systems have been characterized,
either biochemically, structurally or mechanistically. As a
consequence, broad generalizations about the functioning
of these systems are extrapolated from just a few observa-
tions, and therefore a pressing need exists to extend these
characterizations to other transporters.
Furthermore, although the basic features of the
nucleotide-binding sites are understood, less is known
about the binding modes of the translocated substrate
and how this is coupled to the nucleotide state. For
importers, the binding protein has a crucial role — one or
more binding sites might also be present in the transloca-
tion pathway, as observed in the MalFGK2 structure23. For
exporters, particularly multidrug resistance proteins,
the details of the substrate-binding site have yet to be
crystallographically identified18,24.
The high-resolution structural characterization of
eukaryotic ABC transporters, particularly in humans,
remains a fertile area for research, not only for the intrinsic
mechanistic interest but also for the therapeutic poten-
tial that comes from the characterization of substrate-
binding, nucleotide and regulatory sites in these systems.
Furthermore, ABC subunits are not intrinsically special-
ized to function exclusively in transport systems, but are
also integrated into a range of other processes, including
DNA repair and chromosome maintenance74. It has been
proposed in these systems that ATP binding and hydrol-
ysis drives the reversible association and dissociation of
these subunits to act as rings or snaps to tether together
substrates, such as DNA or chromosomes. These mecha-
nistic similarities with transporters are important areas
to explore.
VOluMe 10 | MArCH 2009 | 225
REVIEWS

Mechanistic studies of the translocation reaction of ATP
transporters are complicated by the challenges of work-
ing with membranes and membrane proteins, particularly
when addressing such fundamental questions as the ratio
of ATP molecules hydrolysed per substrate translocated,
the timing between ATP hydrolysis and translocation,
and how substrate binding is coupled to ATP hydrolysis.
This is an area in which single-molecule methods have
great potential to follow the behaviour of individual
transporters without the effects of ensemble averaging.
The goal of these efforts should be to understand in
quantitative detail the molecular and kinetic mecha-
nisms of ABC transporter function and ABC proteins
in general. Not only will such insights be informative
for the development of therapeutically useful agents, but
they will also help us appreciate how one basic engine
can generate the power to change the conformations
of a range of distinct protein systems and thereby
achieve the movement of molecules in, out and
around cells.

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Acknowledgements
We thank A.T. Lee and J.B. Howard for discussions and their
long-term contributions to our research in this area. The sup-
port of the Fulbright Foundation and Jane Coffin Childs
Memorial Funds for Medical Research (O.L.) and National
Institutes of Health grant GM45162 (D.C.R) is gratefully
acknowledged.
DATABASES
Protein Data Bank: http://www.pdb.org/pdb/home/home.do
1H8E | 1L7V | 1M34 | 1Q12 | 2HYD | 2ONJ | 2ONK | 2NQ2 |
2QI9 | 2R6G | 3B5W | 3B5X | 3B5Z | 3B60 | 3D31 | 3DHW
UniProtKB: http://www.uniprot.org
BtuC | BtuD | BtuF | Hi1470 | Hi1471 | LivJ | LivK | MalE | MalF |
MalG | MalK | MetI | MetN | MetQ | ModA | ModB | MsbA |
RbsB | RbsC | Sav1866
FURTHER INFORMATION
Douglas C. Rees’s homepage:
http://www.br.caltech.edu/reesgrp
MolScript: http://www.avatar.se/molscript
Raster3D: http://skuld.bmsc.washington.edu/raster3d
SUPPLEMENTARY INFORMATION
See online article: S1 (box) | S2 (box) | S3 (box)
all liNkS are acTive iN The oNliNe PDf
VOluMe 10 | MArCH 2009 | 227