ETH Library
Mechanistic diversity in
ATP-binding cassette (ABC)
transporters
Review Article
Author(s):
Locher, Kaspar P.
Publication date:
2016
Permanent link:
https://doi.org/10.3929/ethz-b-000117629
Rights / license:
In Copyright - Non-Commercial Use Permitted
Originally published in:
Nature Structural & Molecular Biology 23(6), https://doi.org/10.1038/nsmb.3216
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Mechanistic diversity in ATP-binding cassette (ABC) transporters
Kaspar P. Locher
Institute of Molecular Biology and Biophysics, Department of Biology, ETH Zurich.
Abstract
ABC transporters catalyze transport reactions ranging from high-affinity uptake of micronutrients into
bacteria to the extrusion of cytotoxic compounds from mammalian cells. Crystal structures of ABC
domains and full transporters have provided a framework for formulating reaction mechanisms of
ATP-driven substrate transport, but recent studies suggest a remarkable mechanistic diversity within
the protein family. This review evaluates the differing mechanistic proposals and outlines future
directions to explore ABC transporter-catalyzed reactions.
Introduction
ABC transporters are a family of membrane proteins that mediate diverse, ATP-driven transport
processes. They contain a pair of conserved, cytoplasmic domains termed ATP-binding cassettes
(ABC) or nucleotide-binding domains (NBD). NBDs hydrolyze ATP and drive conformational changes
in the attached transmembrane domains (TMDs), allowing substrates to cross the lipid bilayer of the
membrane either into the cytoplasm (import) or out (export). The domain arrangement and conserved
sequence motifs are summarized in box 1, and representative structures of the different sub-families
of ABC transporters are shown in Fig. 1.
ABC transporters are found in all domains of life. In bacteria, ABC importers are involved in the
uptake of nutrients and micro-nutrients through medium- and high-affinity pathways
1,2
. These
reactions are highly specific and require a periplasmic (or lipid-anchored external) substrate binding
protein
3,4
. Bacterial ABC exporters have diverse roles, including the extrusion of (often lipid-linked)
building blocks required for cell wall assembly
5-7
. Other ABC exporters are poly-specific and
contribute to drug resistance by exporting certain toxic substances
8,9
.
There are 48 distinct human ABC transporters that belong to various sub-families
10
. Most are thought
to be exporters, and some display a remarkable poly-specificity for substrates. Some human ABC
transporters have functions other than substrate translocation, e.g. in regulating potassium channels
(SUR1 and SUR2)
11,12
or acting as an ATP-gated chloride channel (CFTR)
13
. Many human ABC
transporters are bio-medically and clinically relevant: For example, multidrug transporters such as
ABCB1, ABCG2, or ABCC1 protect various organs from toxic insult, but also contribute to drug
resistance of cancer cells
14-17
; In liver, a series of ABC transporters (ABCB11, ABCB4, ABCG5/G8)
catalyzes the generation of bile, with dysfunction of either transporter leading to liver disease
18-22
; The
transporter of antigenic peptides (TAP, a heterodimer of ABCB2 and ABCB3) has an important role in
the adaptive immune response
23,24
. Archaea, yeasts, plants, and human parasites also have ABC
transporters that catalyze vital functions (reviewed in
25-27
).
In addition to the fundamental question of how they translocate large substrates across lipid bilayers,
a detailed structural and mechanistic understanding of ABC transporters may contribute to drug
discovery, for example in the case of multidrug or liver ABC transporters
28-31
.
It has been established that the closed NBD dimer conformation is essential for ATPase activity of full
ABC transporters, because physical NBD separation results in the transporter inactivation
32-34
. Given
the tight interaction between the NBDs and the coupling helices, it is generally accepted that the
conserved power stroke of ABC transporters is the pushing together and pulling apart of the
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2
cytoplasmic ends of the TMDs during the ATP hydrolysis cycle (reviewed in
35-38
). However, recent
structural and mechanistic studies challenged the notion that a single, conserved mechanism can
explain the reactions even within individual sub-families of ABC transporters. This review evaluates
transport mechanisms derived from structures of full transporters as well as from functional,
spectroscopic, and biophysical studies.
Alternating access in ABC importers
Type I ABC importers. Type I ABC importers facilitate medium-affinity uptake of diverse nutrients
including ions, sugars, amino acids, short peptides, and oligosaccharides, into bacteria. The E. coli
maltose transporter has been extensively studied (REFS) and can serve as a mechanistic model
of a
generic mechanism of type 1 ABC importers (Fig. 2a). The transport cycle can be thought to start in
an inward-facing conformation, with substrate-loaded binding protein approaching the external side of
the transporter. There is a possibility of futile ATPase activity at this state, resulting in a "basal
ATPase rate". However, this activity is slow in type 1 ABC importers and often additionally inhibited.
Docking of the substrate binding protein stimulates the basal ATPase rate of the maltose
39
and
alginate transporters
40
, but not of the molybdate/tungstate transporters
32,41
. The key conformational
transition during the transport cycle (state 1 to 2) is the closing of the NBDs, which pushes the
coupling helices towards each other and converts the TMDs to an outward-facing conformation. This
generates a tunnel from the attached binding protein to a low-affinity substrate-binding pocket in the
translocation pathway, halfway across the membrane. In the maltose transporter, two effects were
found to ensure the release of substrate from the binding protein: First, the binding pocket is slightly
distorted. Second, periplasmic TMD loops (in particular a "scoop loop") cause a partial steric clash
with bound substrate
42
. Although the affinity of maltose to the temporary binding site between the
TMDs is only moderate (Kd in the millimolar range), the substrate populates this pocket because the
local concentration in the tunnel is at least 2 orders of magnitude above the dissociation constant. In
the subsequent, irreversible step (state 2 to 3) ATP is hydrolyzed and inorganic phosphate released.
This causes the NBD dimer to open, pulling the coupling helices outwards and triggering the
conversion to an inward-facing conformation. The substrate can now dissociate into the cytoplasm.
Finally, the exchange of ADP for ATP resets the system (state 3 to 1).
There are mechanistic variations of the theme in certain type 1 ABC importers. Because of their role
in medium-affinity pathways, efficient coupling of ATP hydrolysis to transport is important, and the
observed basal ATPase activities are generally low. While some transporters such as OpuA have
strict coupling of 2 ATP per transport cycle
43
, additional regulatory strategies have been observed for
other transporters in the form of trans-inhibition
32,33,44
or the binding of regulatory proteins
45
.
Type 2 ABC importers. These are generally part of high-affinity uptake pathways for metal chelates
including heme and other iron-containing complexes, and cobalamin (reviewd in
46,47
). These
substrates are not only larger and more hydrophobic than those of type I ABC importers, but available
at low concentrations only. The mechanism proposed for the E. coli vitamin B12 transporter BtuCD-F
can serve as a model for other type 2 ABC importers. Unlike observed in the maltose transporter,
there is no measurable affinity (and thus no substrate binding pocket) for B12 in the TMDs of BtuCD-
F. The obtained crystal structures revealed three gate regions in the transmembrane BtuC subunits,
two on the cytoplasmic side (cyto-gates 1 and 2) and one on the periplasmic side (peri-gate)
48,49
. The
key difference to type 1 ABC importers is that in BtuCD-F, ATP binding and hydrolysis does not
generate an inward-facing conformation by itself. Rather, the two cytoplasmic gates separate the
cytoplasm from the central translocation cavity both in the ATP-bound and nucleotide-free states, as
long as transport substrate is absent. Figure 3b shows a schematic of the transport mechanism
deduced from structural, biochemical, and spectroscopic data
48-52
. The cycle can be thought to start in
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3
an ATP-bound state (state 1) with a closed NBD dimer conformation, and coupling helices pushed
together, which causes cyto-gate 2 to close. Because the peri-gate is open, state 1 is an outward-
facing conformation. Futile ATPase activity (arrows to the left) has been observed in all type 2 ABC
importers studied to date, although the measured rates are very low and the loss of cytoplasmic ATP
apparently manageable for the cell. State 2 is reached upon docking of the substrate binding protein
and the release of substrate into a hydrophobic ("teflon") cavity with no measurable affinity. As in type
1 importers, the high-affinity pocket in the substrate binding protein is distorted upon docking, and
loops of the TMD cause a steric clash, "scooping" the substrate from its pocket and enclosing it in the
translocation pathway between the TMDs. In BtuCD, trapping of substrate in this cavity could only be
demonstrated in liposomes. In detergent, the substrate escapes from the cavity, probably due to
increased protein dynamics or competition for binding with detergent molecules. The following,
irreversible step (state 2 to 3) is the hydrolysis of ATP and the release of phosphate, which requires
(at least partial) opening of the NBD dimer, pulling the coupling helices apart and opening cyto-gate 2.
Due to the size of the trapped transport substrate, cyto-gate 1 is now unable to close, which probably
causes a strained conformation and pressure from the sides onto the substrate. This resembles a
peristaltic force and may contribute to substrate dissociation into the cytoplasm. Upon substrate
release, BtuCD relaxes into an asymmetric conformation (state 4) that does not feature a central
cavity anymore, because both cyto-gate 1 and the peri-gate are now closed
51
. This state is very stable
in vitro and probably probably prevents potential ion leakage during the subsequent re-setting of the
system. The asymmetric conformation of state 4 appears unique to type 2 importers because the
structural elements involved are not present in type 1 importers. Notably, the mechanistic proposal
does not assign any role to the BtuCD conformation first observed, an apo-state with outward-facing
TMDs
53
. Although such a conformation may transiently occur in the cell (before ADP is replaced by
ATP) and contribute to futile ATP hydrolysis, it is not essential to formulate a productive transport
cycle. Another important point is that nucleotide-bound BtuCD with a closed NBD dimer could only be
stably trapped and crystallized upon introduction of a disulfide cross-link that covalently linked the D-
loops of the two NBDs.
Distinct mechanistic proposals for B-family ABC exporters
Despite a number of high-resolution structures reported, no common mechanism has been
established for B-family ABC exporters. One problem is that most structures do not reveal bound
substrates, a consequence of their hydrophobicity and low binding affinity in detergent solution.
Another issue is that when removed from the membrane and in the absence of ATP (or ATP-ADP
mixtures reflecting average cytoplasmic concentrations), B-family ABC exporters are prone to
adopting inward-facing conformations with occasionally very large separations of the NBDs. When
bound to nucleotides, the conformations tend to show a smaller separation of the NBDs as well as
occluded or outward-facing conformations. Figure 3 shows a selection of structures of B-family ABC
exporters, demonstrating the wide conformational range.
Early mechanistic proposals stated that alternating access in B-family ABC exporters is reached by
converting the observed, inward-facing conformations to outward-facing states with bound ATP.
However, several crystal structures are inconsistent with this simplified view
54-58
. The relevance of the
observed inward-facing conformations has therefore been controversially discussed (see box 2), and
the discussion is not simply about semantics. The critical issue is whether an observed structure
reflects a functionally relevant state. If it is concluded that a conformation is not physiologically
relevant (as were the inward-facing conformation of the LLO flippase PglK), there would be no point in
attempting to dock a transport substrates into the observed cavities. The two extreme views outlined
in box 2 could be reconciled by viewing B-family ABC exporters as springs, whose architecture works
to push their NBDs apart. In cellular membranes, the combined effects of lipids, ATP, and substrate
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4
would allow the transporters to reach outward-facing conformations. Inward-facing conformations
might reflect physiological states if they provide substrate binding sites or may occur transiently
because the NBD dimer needs to release the ATP hydrolysis products. Inward-facing conformations
without NBD contact or substrate binding pockets are likely unphysiological.
As was observed for ABC importers, substrates and inhibitors can modulate the ATPase activity of B-
family ABC exporters. The molecular basis of this phenomenon is unknown, but stimulation or
inhibition is often used to identify specific interactions. Two distinct mechanisms of B-family ABC
exporters are shown in Figure 4.
Alternating access. An essential component of this mechanistic proposal (Fig. 4a) is the binding of
transport substrate to an inward-facing pocket of the TMDs (state 1). The relevant binding site may
not only be accessible from the cytoplasm, but also from the inner leaflet of the lipid bilayer. Highly
hydrophobic substrates (drugs, phospholipids) could thus be directly recruited from within the
membrane. The inward-facing conformation is linked to an open NBD conformation. As for ABC
importers, futile ATP hydrolysis may occur in the absence of substrate, albeit at a slower rate than
when substrate is bound. For poly-specific transporters (including the multidrug transporter ABCB1), it
is unclear if there is only one binding pocket
59
, as some findings suggest multiple sites
60,61
. In the case
of TAP, it was concluded that the N- and C-termini of all transported peptides are firmly bound,
whereas the central aminoacids may only weakly interact with the transporter
62
. Once a substrate is
recruited, the transporter probably converts to an occluded conformation (state 1 to 2), with ATP
bound between closed NBDs, but without access to the central cavity from either side of the
membrane. Such a conformation was observed in the structures of AMPPNP-bound McjD
63
and
ATPγS-bound PCAT
57
, albeit without bound substrate. It is unclear if the occluded conformation is a
mandatory intermediate. The following step (state 2 to 3) reflects the conversion to the outward-facing
conformation, allowing substrate release into the external medium or into the outer leaflet of the lipid
bilayer. Given that there is a re-arrangement of the TM helices from states 1 to 3, with different sets of
helices surrounding the translocation pathway and forming the two external wings (Fig. 4), there is a
change in the cavity surface surrounding the substrate. In the AMPPNP-bound, outward-facing
Sav1866 structure, a rather hydrophilic cavity was observed, suggesting that a chemical mismatch to
the hydrophobic drugs could be driving substrate release
64
. This notion was in line with observations
that the apparent affinity of multidrug ABC transporters for substrates was lower from the external
than from the cytoplasmic side
65
. If a substrate is highly hydrophobic (such as in the case of certain
tumor drugs or phospholipids), it is likely to re-partition into the membrane and may only remain in the
external medium when bound to a carrier protein (e.g. BSA) and swept away in the blood stream, or if
stabilized in a mixed micelle such as in bile. In the final step (state 3 to 1), hydrolysis of ATP and the
release of inorganic phosphate drives the NBDs apart and, by transmitting this motion to the TMDs
via the coupling helices, converts the outward-facing into an inward-facing conformation. This step is
irreversible given the large amount of energy gained from the hydrolysis of ATP (approaching 50kJ
mol
-1
for one ATP). It is unclear if the hydrolysis of two ATP molecules is simultaneous or consecutive,
nor is it certain that both ATP molecules are hydrolyzed during every transport cycle (see below).
"
Outward-only mechanism. This mechanism was proposed for the LLO flippase PglK
66
(Fig. 4b). As
its name implies, the outward-only mechanism does not invoke an inward-facing cavity to interact with
the substrate. Although two distinct inward-facing conformations were determined for PglK, the
observed cavities did not appear to contribute to the interaction with the substrate. The cycle of the
outward-only mechanism can be thought to start in an ATP-bound state and an outward-facing TMD
conformation (state 1). Whereas the lipidic polyprenyl tail of the substrate probably remains attached
to the lipid-facing surface of the transporter, the pyrophosphate-glycan head-group is proposed to be
transferred directly into the outward-facing cavity, where strong, electrostatic interactions between
