Cent. Eur. J. Biol.• 6(5) • 2011 • 785-801 DOI: 10.2478/s11535-011-0054-4

Central European Journal of Biology

ATP binding cassette systems: structures, mechanisms, and functions

Review

Anke Licht*, Erwin Schneider*

Department of Biology, Division of Microbial Physiology, Humboldt University of Berlin, D-10115 Berlin, Germany

Received 31 March 2011; Accepted 02 June 2011

Abstract: ATP-binding cassette (ABC) systems are found in all three domains of life and in some giant viruses and form one of the largest protein superfamilies. Most family members are transport proteins that couple the free energy of ATP hydrolysis to the translocation of solutes across a biological membrane. The energizing module is also used to drive non-transport processes associated, e.g., with DNA repair and protein translation. Many ABC proteins are of considerable medical importance. In humans, dysfunction of at least eighteen out of 49 ABC transporters is associated with disease, such as , Tangier disease, adrenoleukodystrophy or Stargardt’s macular degeneration. In prokaryotes, ABC proteins confer resistance to , secrete virulence factors and envelope components, or mediate the uptake of a large variety of nutrients. Canonical ABC transporters share a common structural organization comprising two transmembrane domains (TMDs) that form the translocation pore and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. In this Mini-Review, we summarize recent structural and biochemical data obtained from both prokaryotic and eukaryotic model systems.

Keywords: ATP-binding cassette • Protein superfamily • Transport proteins • P-glycoprotein • Maltose transporter • ECF transporter • Prokaryotes • Eukaryotes • Disease

© Versita Sp. z o.o.

1. Introduction size, the highest number of ABC systems is found in [1]. ATP-binding cassette (ABC) systems are found in all Canonical ABC transporters share a common three domains of life (bacteria, archaea and eukarya) structural organization comprising two transmembrane and some giant viruses and form one of the largest domains (TMDs) that form the translocation pathway protein superfamilies [1]. Most family members are and two nucleotide-binding domains (NBDs) that bind transport proteins that couple the free energy of ATP and hydrolyze ATP. Based on sequence comparison, hydrolysis to the translocation of solutes across a three classes of ABC systems that were probably biological membrane. However, the energizing module present in the last common ancestor of archaea, bacteria is also used to drive non-transport processes. The and eukarya have been proposed: class 1 comprises human genome includes 49 genes for ABC transporters transporters with fused TMDs and NBDs (exporters), in seven subfamilies (designated A-G), at least eighteen class 2 includes non-transport ABCs lacking TMDs and of which, when mutated, cause disease [2], while the class 3, (which is absent in eukaryotes) includes mainly genome of the gut bacterium Escherichia coli strain K-12 transporters with NBDs and TMDs formed by separate was predicted to encode 71 discrete ABC (transport) polypeptide chains (canonical importers) and some systems [3] (http://www1.pasteur.fr/recherche/unites/ bacterial exporters (Figure 1) (reviewed in [1]). pmtg/abc/database.iphtml). Substrates transported by ABC transporters are Plant genomes code for a high number of ABC diverse, such as sugars, amino acids, peptides, vitamins, systems with more than 120 in both Arabidopsis thaliana ions, hormones, xenobiotics, antimicrobial drugs, and Oryza sativa (rice) [4]. Yet, in relation to genome chemotherapeutic agents, and even large polypeptides,

* E-mail: [email protected] ; [email protected] 785 ATP binding cassette systems: structures, mechanisms, and functions

Figure 1. Modular organization of ATP-binding cassette systems. Members of the ABC superfamily are classified into exporters, importers and non-transporting proteins. Exporters and canonical importers consist of two transmembrane domains (TMD, in green) and two nucleotide-binding domains (NBD, in purple). In exporters, the substrate (filled black circles) might enter the translocation pore from the cytoplasmic side or from within the membrane, depending on its chemical nature. As a representative, the crystal structure of mouse P-glycoprotein (MDR1) (PDB code 3G5U) is shown. MDR1 is a full transporter with all four domains fused in a single polypeptide chain (see also Figure 3A). Canonical ABC importers require an extracytoplasmic solute receptor (in orange). As a representative, the structure of the molybdate/tungstate transporter of Archaeoglobus fulgidus is shown, consisting of a homodimer each of the TMD, ModB (green), the NBD, ModC (violet), and the receptor, ModA (orange) (PDB code 2ONK). ECF (energy coupling factor) type ABC importers consist of a substrate-specific transmembrane domain (S component), a small transmembrane domain (T component), and two copies of a nucleotide binding domain (NBD, also termed A and A’ components). The structure of the S-component RibU of the riboflavin transporter of Staphylococcus aureus with bound substrate (yellow, space-fill representation) is shown (PDB code 3P5N). Non-transporting ABC proteins consist of two fused NBD dimers and additional domains (in light yellow) but lack TMDs. As a representative, the structure of the catalytic domain of the DNA repair protein Rad50 of Pyrococcus furiosus is depicted in complex with two ATP molecules (red) (PDB code 1F2U). N-and C-terminal domains are coloured purple and cyan, respectively. Structures are not necessarily drawn to scale.

linking these proteins to various cellular functions to sense the γ–phosphate, bind the necessary divalent that range from energy supply to osmoregulation, cation (usually Mg2+) and attack water [6]. The LSGGQ detoxification and virulence. motif interacts with the γ–phosphate and is involved in ATP In ABC transporters, substrate specificity is hydrolysis as known from the arginine finger observed in accomplished by the TMDs, which display basically no other P-loop ATPases [7]. In the active NBD dimer, ATP is sequence homologies and feature varying numbers of sandwiched between the Walker A motif of one monomer transmembrane helices (5-10) among different systems. and the LSGGQ motif of the opposing monomer [6] On the contrary, the ability to bind and hydrolyze ATP, thus (Figure 2C). providing the energy stroke for substrate translocation or As a unique feature of ABC transporters, the so- non-transport processes, is a common feature of the NBDs, called ‘coupling helices’ or ‘EAA’ motifs in canonical which share several conserved motifs in all ABC systems importers, localized to the last cytoplasmic loop of the (Figure 2A). With regard to three-dimensional structures, importer TMDs, and the first and last cytoplasmic loops NBDs can be subdivided in a RecA-like subdomain, of exporter TMDs, are part of the NBD–TMD interface. containing the conserved Walker A (also known as ‘P-loop’) The ‘coupling helices’ are architecturally conserved and B motifs which contribute substantially to nucleotide elements, which interact with the Q-loops to couple binding, and an α-helical subdomain encompassing the binding and hydrolysis of ATP at the NBDs to additional conserved motifs. Among these motifs are the conformational changes in the TMDs. Q-loop (containing a conserved glutamine residue) and ABC exporters are mostly NBD-TMD fusion proteins the signature sequence (‘LSGGQ’) by which members that form either homo- or heterodimers (‘half transporter’) of the family can be identified [5] (Figure 2B). The or all four domains are located on one polypeptide chain Q-loop, located at the interface to the TMDs, is known (Figure 3A).

786 A. Licht, E. Schneider

Figure 2. Conserved motifs of NBDs. A. Linear representation of an NBD, with assignment of the main functional sites. Conserved sequences are in parentheses, using the one letter code for amino acids (x, any residue; h, hydrophobic residues). Motifs involved in nucleotide binding are given in red. B. Structure of an NBD monomer (ArtP of Geobacillus stearothermophilus, PDB code 2OUK) indicating the

localization of conserved motifs as shown in (A). C. Structure of an NBD dimer (MalK2 of E. coli, PDB code 1Q12; regulatory C-terminal domains are omitted) with two ATP molecules (blue, space fill representation) sandwiched between the Walker A motif of one monomer (A chain, in yellow) and the ABC signature of the opposing monomer (B chain, in light blue).

Figure 3. Diversity in molecular architecture of ABC transporters. A. ABC exporters might be homo- (Sav1866) or heterodimers (TAP1/TAP2) of a TMD-NBD fusion protein. Alternatively, all four domains can be fused into a single polypeptide chain (MDR1), e.g. in the order of TMD1-NBD1-TMD2-NBD2 or, as in case of CFTR, an additional regulatory domain (in yellow) is connecting NBD1 with TMD2. B. Canonical ABC importers can exist in all possible combinations of separately expressed or fused TMDs and NBDs. The extracellular solute receptors are either soluble proteins, lipid-anchored, membrane-associated via an N-terminal transmembrane α-helix or fused to a TMD (not depicted) [8]. Substrates transported by the indicated systems are given in parentheses. Colour code as in Figure 1.

Canonical (class 3) ABC import systems, hitherto architecture is diverse, i.e. all possible combinations exist confined to prokaryotes, are dependent on high-affinity, (Figure 3B). Canonical importers have been further sub- extracellular (or periplasmic) solute binding proteins divided into type I and type II based on structural and (SBP) (also called receptors) (Figure 1). Their domain biochemical evidence (see section 4 for details).

787 ATP binding cassette systems: structures, mechanisms, and functions

In contrast, ECF (‘energy coupling factor’) importers, long chain fatty acid transporter ALDP (ABCD1) cause also widespread among prokaryotes, lack a typical adrenoleukodystrophy, and defective MDR3 and BSEP SBP and are composed of an energy coupling module (ABCB11) mediating the export of phosphatidylcholine comprising one conserved TMD (T-component) and two and bile acids, respectively, are associated with liver NBDs (A-components), which require an additional small disease (overview: [13]). integral membrane protein (S-component) to capture ABC transporters are also involved in lipid specific substrates (micronutrients like vitamins and translocation in bacteria. The export of lipopolysaccharide certain transition metals) (reviewed in [8]) (Figure 1). (LPS), an essential component of the outer membrane Currently, the transporter classification (TC) system, of Gram-negative bacteria, consisting of a lipid A moiety which is analogous to the Enzyme Commission system (also known as enterotoxin) linked to the O-antigen via for classification of enzymes but incorporates both a core oligosaccharide is mediated by the lipid A flippase functional and phylogenetic information, includes 34 MsbA and the complete LPS exporter machinery, Lpt. subfamilies of ABC importers and 53 subfamilies of ABC Whereas the homodimeric MsbA transports modified exporters within family 3.A.1. (http://www.tcdb.org). lipid A from the cytoplasmic to the periplasmic side of the Crystal structures are available for numerous inner membrane, the O-antigen-linked complex is then isolated NBDs (summarized in [3]), three ABC exporters, extracted from the outer leaflet of the inner membrane eight canonical importers, including four with bound by the ABC exporter LptBFG-C and shuttled to the outer receptor [9,10], an S- component [11] and several non- membrane by the action of the periplasmic chaperone transporting ABC proteins [1,12]. Examples are shown LptA and the outer membrane complex LptDE [18]. in Figure 1. The human heterodimeric transporter associated In this Mini-Review, we provide the reader, in with antigen processing (TAP), composed of ABCB2 particular the non-specialist in the field, with an (TAP1) and ABCB3 (TAP2) translocates peptides overview on the current knowledge on the ABC protein derived from the proteasomal degradation system into superfamily. Following the classification system based the lumen of the endoplasmic reticulum for loading on phylogenetic analysis, we summarize recent onto major histocompatibility complex (MHC) class I structural and functional data on model systems molecules. Hence, the TAP exporter functions in MHC from both prokaryotes and eukaryotes that belong to class I antigen presentation and is suggested to be exporters, importers, and non-transporting systems. involved in peptide presentation to MHC class II in For further details, the reader is referred to a number of dendritic cells [19]. reviews published in recent years [1,6,8,9,13-16]. Remarkably, a wide range of ABC exporters characterized to date extrude a broad range of chemically diverse drugs, a phenomenon designated 2. ABC exporters as multidrug resistance (MDR). Thus, ABC exporters have gained considerable attention due to their The ubiquitously found ABC exporters mediate the contribution to the resistance of cells to antibiotics and translocation of chemicals from the cytoplasm or the chemotherapeutic agents. Within the human genome, inner leaflet of the lipid bilayer to the exterior ofthe at least three ABC transporters have been identified as cell or into the lumen of organelles (e.g. mitochondria, MDR pumps: P-glycoprotein (MDR1, P-gp), multidrug peroxisomes, vacuoles and the endoplasmic reticulum) resistance protein 1 (MRP1), and breast cancer [17]. The substrates differ enormously in size and resistance protein (BCRP), belonging to subfamilies chemical nature, including metabolic intermediates, ABCB, ABCC and ABCG, respectively [16]. Of these, lipids, sterols, antibiotics, proteins and peptides, small P-glycoprotein was the first drug efflux pump described drug molecules or complex carbohydrate polymers of and is the best-studied MDR exporter to date. In general, the bacterial cell wall [1] (Table 1). For example, several P-glycoprotein is synthesized at varying levels in every mammalian ABC transporters contribute to the control human tissue, where it is involved in the maintenance of cellular lipid export processes and have been linked of cholesterol distribution across the leaflets of the to severe genetic diseases. Thus, defects in the human plasma membrane. Additionally, this system mediates sterol transporters Sterolin1 and 2 (ABCG5/ABCG8) the efflux of a plethora of hydrophobic drugs, such as cause (see ‘Glossary’ for explanation vinca alkaloids like the cell division inhibitor vinblastine of medical terms used in this article), a rare inherited or anthracyclines, and the DNA-intercalating agents disorder, a functional loss of human ABCA1 involved daunorubicin and doxorubicin [20]. In the past years, in biogenesis of high density lipoprotein (HDL) has numerous reports were published that focused on been linked to Tangier disease, defects in the very the structural and biochemical characterization of

788 A. Licht, E. Schneider

Suggested Transporter Transported substrate(s) Properties, function, disease reading

Bacteria HlyBDTolC haemolysin virulence factor of some E. coli strains [72] LmrA multiple drugs mode of resistance [73] DrrAB doxorubicin, daunorubicin mode of resistance [74] MsbA, LptFGB Precursors of lipopolysaccharide component of bacterial outer membrane [18] LolCDE Lipoproteins component of bacterial outer membrane [75] Yeast Ste6 a-factor mating pheromone of Saccharomyces cerevisiae [76] Pdr5 multiple drugs mode of resistance [26] Plants AtABCB1 auxin (indol-3-acetic acid) phytohormone [15] AtABCC1 glutathione conjugates detoxification [15] ATABCG36 Cd2+ detoxification [15] Human TAP1/TAP2 (ABCB2/ABCB3) antigenic peptides transport to the ER [19] P-glycoprotein (MDR1; ABCB1) membrane lipids amplification in certain cancer cells causes multiresistance [77] against chemotherapeutic drugs ALDP (ABCD1) long chain fatty acids defect causes hereditary disease adrenoleukodystrophy [78] ABCR (ABCA4) retinal defect causes hereditary disease Stargardt’s macular [79] degeneration ABC1 (ABCA1) cholesterol defect causes hereditary Tangier disease [80] CFTR (ABCC7) chloride ions channel; maintenance of ion currents (mutations cause most [81] common hereditary disease cystic fibrosis) SUR1/SUR2 none sulfonylurea receptors, NBDs of complex potassium channel [31] (ABCC8/ABCC9)

Table 1. Examples of ABC exporters.

drug binding sites in order to elucidate the molecular be drawn. All structures share a conserved architectural mechanism by which a single protein can specifically arrangement with a core of 12 TM helices and two recognize and extrude a huge diversity of substrates NBDs. An additional N-terminal domain (TMD0), most (reviewed in [16,21]). Altogether, multidrug exporters likely involved in regulatory functions only, is observed in seem to contain a large, hydrophobic binding pocket several human homologues, especially within the ABCC that accommodates several, partially overlapping, sub- subfamily but is also known from TAP1/TAP2 [24]. In sites with varying preferences for different substrates. each ‘half transporter’ (see Figure 3A), TM helices Especially, in the case of P-glycoprotein, a general 2-6 are extended significantly beyond the membrane substrate structure of two or three electron donors with a boundary with an average length of almost 70 Å. In fixed spatial separation was suggested (for discussion, contrast to ABC importers (see below), exporters see [1]). exhibit a substantial domain intertwining of the TMDs However, for a full understanding of the mode forming a wing-like structure with each lobe consisting of action, high resolution tertiary structures of ABC of TM helices 1-3 and 6 from one protomer and helices exporters are indispensable. To date, only the X-ray 4-5 from the opposite. As a consequence, the domain crystallographic structures of one eukaryotic and two swapping continues through the TMD-NBD interface prokaryotic ABC exporters have been solved: the mouse such, that the coupling helix 2 connecting TM helices multidrug exporter MDR1A [10], the lipid flippase MsbA 4 and 5 crosses over to contact exclusively the NBD from E. coli [22], and the multidrug exporter Sav1866 of the other monomer, while coupling helix 1 between from Staphylococcus aureus [23]. Although resolution is TM helices 2 and 3 predominantly interacts with the own only between 3 - > 5 Å, the following conclusions can NBD. Thereby, each coupling helix contacts residues

789 ATP binding cassette systems: structures, mechanisms, and functions

both of the Q- loop and within a conserved motif to the inward-facing conformation, thus completing the (defined as X-loop) located immediately N-terminal transport cycle [27] (Figure 4A). Certainly, more X-ray from to the LSGGQ sequence [25]. This finding is structures of the same protein in both the inward and consistent with biochemical evidence obtained with outward facing conformation at resolutions < 3 Å are human TAP1/2 transporter, human P-glycoprotein, desirable to refine the model at atomic resolution. the Cystic Fibrosis Transmembrane Conductance It should also be noted that the stoichiometry of Regulator (CFTR, ABCC7), and the yeast multidrug nucleotides actually hydrolyzed per translocation event ABC exporter Yor1p (reviewed in [6]). Together, these is still under debate. Whereas the ATP switch model results suggest a similar architecture in other eukaryotic as summarized above predicts ATP hydrolysis at both ABC exporters, at least those belonging to subfamilies sites per cycle, the observed ATPase site asymmetry in B and C. However, the functional P-glycoprotein several ABC transporters, such as human CFTR, TAP homologue Pdr5p conferring pleiotropic drug resistance and yeast Pdr5, argue in favor of ATP hydrolysis at only (PDR) in yeast differs significantly in sequence and one site as being sufficient for restoration of the inward- architecture. Consisting of degenerated NBDs and an facing TMD conformation (reviewed in [14]). inverted topology (NBD-TMD-NBD-TMD), Pdr5p is a Sav1866 is a close homologue of the bacterial ABC non-canonical ABC transporter that exhibits only basal exporters MsbA and the multidrug exporter LmrA from ATPase activity, which, in contrast to MDR1, is not Lactococcus lactis [28]. The purified protein displayed stimulated by the tested substrates [26]. drug-stimulated ATPase activity and was demonstrated In the structure of the bacterial Sav1866 exporter, to transport a model drug in proteoliposomes [29]. the NBDs form a head-to-tail closed dimer with two Hence, Sav1866 can likely be used as a reliable molecules of AMP-PNP, a non-hydrolysable ATP structural model of other ABC exporters, including analog, bound at the interface. In contrast to the P-gp. To understand and therefore to prevent the MDR P-glycoprotein structure (see also Figure 1), the TMDs phenotype of human ABC exporters there is a need to are arranged in an outward-facing manner, containing identify specific structural key features in prokaryotic a central cavity that is open to the extracellular space ABC efflux systems to synthesize effective inhibitors [25]. The large substrate-binding cavity is mainly interfering with substrate binding and/or transport hydrophilic and therefore was assumed to exhibit a low activity. However, the prokaryotic ABC exporters form affinity for the hydrophobic ligands. This is supported functional homodimers, whereas most eukaryotic ABC by biochemical studies, which demonstrated a drastic transporters like human P-gp are ‘full transporters’ decrease in substrate binding affinity upon ATP-binding comprising a single polypeptide chain (Figure 3A). This to the NBDs (reviewed in [27]). In contrast, the inward- raises the question, whether structures of prokaryotic facing structure of P-gp in complex with cyclic peptide ABC exporters can in general serve as homology models inhibitors revealed interactions of the substrate with for eukaryotic members of the family to circumvent hydrophobic and aromatic residues of the internal cavity difficulties in isolating and crystallizing membrane [10]. Based on these and other findings, the following transport proteins, e.g. from mammalian sources. ‘switch’ model of ABC exporters arose (discussed in more detail in [6]; further references therein): in the resting state, the transporter is in an inward-facing 3. Non-canonical human ABC systems conformation with a high-affinity substrate-binding site exposed to the cytoplasmic side and the inner leaflet Although phylogenetically belonging to subfamily of the membrane. Binding of substrate occurs at this ABCC, both the CFTR and SUR1/SUR2 proteins stage independent of nucleotides as observed in the are not canonical ABC exporters. CFTR (Cystic structure of mouse P-glycoprotein [10]. Subsequent Fibrosis Transmembrane Regulator) (ABCC7) is a binding of ATP to the NBDs converts the transporter cAMP-activated, ATP-gated chloride ion channel, not to an outward-facing conformation. In vivo, due to a transporter, that when mutated causes the most the high intracellular ATP concentration, ATP might common hereditary disease cystic fibrosis. In the always be bound to the NBDs and thus, transition of vast majority of cases, the defective protein carries a the transporter to outward-facing would be triggered by deletion of phenylalanine-508 (F508), causing retention substrate binding. Eventually, extrusion of the substrate of ΔF508-CFTR in the endoplasmic reticulum that leads could occur as a consequence of decreased binding to the absence of CFTR chloride ion channels in the affinity caused by changes in specific residue contacts plasma membrane. In CFTR, the first NBD is connected between the protein and substrate [10]. ATP-hydrolysis to the second transmembrane domain by a regulatory and subsequent release of ADP switch the transporter “R” domain (see Figure 3A). Unlike a canonical ABC

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system, ATP is hydrolyzed only at the C-terminal NBD2 Canonical ABC importers mediate the uptake of thereby energizing channel closing while ATP-binding to nutrients, osmoprotectants, various growth factors or both, NBD1 and NBD2, results in channel opening with trace elements (reviewed in [8]) (see also Table 2). a massive chloride ion efflux [30]. For example, a large number of canonical importers The sulfonylurea receptors SUR1 (ABCC8) and act as uptake systems for a diverse range of mostly plant- SUR2 (ABCC9) are ‘full transporters’ but lacking and fungi-derived carbohydrates to be utilized as carbon a transport function, to which potassium-selective and energy sources. The best-characterized member membrane proteins are peripherally attached to is the maltose/maltodextrin transporter MalEFGK2 of assemble ATP-sensitive potassium (KATP) channels E. coli, enabling the facultative anaerobe to feed on found in neurons and endocrine cells. Whereas ATP- degradation products of starch or glycogen of their hosts’ binding stimulates channel closure, Mg2+/ATP-binding diet at the entry of the large intestine. The receptor, and/or hydrolysis in the nucleotide-binding domains of MalE, binds maltose and maltooligosaccharides up to

SUR induces channel opening. Thus, the KATP channel seven glucose units and delivers them to the MalFGK2 monitors the energy balance within the cell. Mutations complex for translocation to the cytoplasm. Two crystal in SUR1 have also been associated with non-insulin- structures of the maltose transporter, representing the dependent diabetes mellitus type II, while a defective apo-state [36] and a catalytic intermediate with bound SUR2 protein is linked to dilated cardiomyopathy [31]. ATP and MalE [37], have contributed substantially to the current transporter model (see below). The latter structure together with biochemical evidence [38,39] 4. ABC importers also revealed a crucial role of a unique extracellular peptide loop of the TMD MalF in contacting MalE and

As already addressed, ABC importers - canonical (class communicating substrate availability to the NBDs, MalK2 3) and ECF systems – are hitherto exclusively found in (see [40] and further references therein). prokaryotes. However, in recent times, experimental Prokaryotes can also utilize amino acids as carbon evidence has accumulated suggesting that distinct and/or nitrogen sources. While a few ABC import ABC systems in plants, yeast and protozoa transport systems accept a wide range of amino acids, most substrates across the plasma membrane into cells. of the transporters are rather specific for chemically closely related compounds such as hydrophobic or polar 4.1. Canonical (binding protein-dependent) amino acids. Of the latter, the histidine-lysine/arginine/

ABC importers ornithine transporter, HisJ/LAO-QMP2, of Salmonella Canonical ABC importers are dependent on high-affinity enterica serovar Typhimurium represents the prototype solute-binding proteins (SBPs) or receptors for function. [41,42]. The ABC subunit of this system, HisP, was the SBPs generally consist of two symmetrical lobes or first NBD of which the crystal structure was solved [43]. domains, which both display an α-/β-fold, connected by a The heterodimeric TMDs, HisQ and HisM, interact with hinge region [32] (Figure 1). Substrate binding causes a two SBPs, HisJ and LAO, which exhibit high affinities in rotation of these lobes towards each other, rendering the the nanomolar range for histidine, and lysine/arginine/ central cleft inaccessible from the aqueous surrounding ornithine, respectively, but bind the preferred substrate(s) (‘Venus-flytrap’-model). Based on more than 120 three- of the other with about 10-fold lower affinities (see [8] for dimensional structures, a classification of SBPs into six review). clusters was recently proposed [32]. The uptake of glutamate and glutamine SBPs bind their substrates with high specificity and as primary nitrogen sources from the host high affinities, displaying dissociation constants typically has been implicated in bacterial virulence of ranging from 0.01-10 μM [32]. Since prokaryotes usually several pathogens like Neisseria meningitidis, thrive in a nutrient-limited environment, capture and S. Typhimurium or group B streptococci (reviewed in accumulation of substrate in proximity to the transporter [8]). is considered as main function of SBPs. However, The maintenance of cell volume and turgor pressure as these proteins are also indispensable at very high in prokaryotes requires the uptake of osmotically active substrate concentrations, they play an important compounds in the cytoplasm, such as potassium ions functional role in the catalytic cycle of the transporter and so-called ‘compatible solutes’ [44]. The best- [33,34]. characterized ABC transporter mediating the uptake Some SBPs are involved in bacterial chemotaxis by of the compatible solutes glycine-betaine and proline interacting with distinct chemoreceptors resulting in a as a response to high-osmolarity growth conditions is movement towards an attractant [35]. the OpuA system of L. lactis. This ABC transporter is

791 ATP binding cassette systems: structures, mechanisms, and functions

composed of an unusual transmembrane subunit to facing, (resting) state with contact to the cognate SBP. which a solute-binding domain is fused, OpuAB, and an Furthermore, due to the high (mM) concentration of ATP-binding subunit, OpuAA, with a C-terminal domain ATP in the bacterial cytoplasm, the resting state of the that functions as a sensor for intracytoplasmic ionic transporter is assumed to have ATP bound to the NBDs strength [45]. which, where analysed, exhibit dissociation constants Of the canonical ABC importers mentioned, the for ATP in the µM range [5]. Upon binding of substrate, enterobacterial maltose and histidine transporters, the SBP is shifted to its closed conformation, which in turn glycine-betaine transporter as well as the homodimeric promotes a tight association of the NBDs, resulting in

vitamin B12 transporter BtuF(CD)2 of E. coli, the first opening of the transport pore to the extracellular side full ABC transporter of which the crystal structure was (‘outward-facing’). Concomitantly, SBP is pushed solved [46], are model systems for the study of ABC towards its open conformation, the substrate becomes transporters’ structure and function. To date, eight released and enters the pore. At this stage, SBP forms prokaryotic ABC importers have been crystallized, three a stable complex with the transporter. Subsequently, of which in complex with their cognate SBP (reviewed hydrolysis of both ATP molecules which might occur in [9]). sequentially, allows release of the substrate to the Based on structural and biochemical studies, cytoplasm. Eventually, dissociation of phosphate and canonical ABC importers may be subdivided into ADP, and rebinding of ATP would restore the resting types I and II [47]. The first type comprises importers state. featuring a transmembrane core of 10-14 helices, In contrast to type I importers, structures of the like the above mentioned maltose and histidine larger homodimeric type II ABC importers [46,52,53] transporters. Type II importers on the other hand possess a densely packed TMD architecture display larger transmembrane domains with up to 20 of ten transmembrane helices per TMD with a helices and specificity for metal chelates, heme, and central translocation pore in between. The latter is

vitamin B12. Thus, in contrast to ABC exporters, the considerably smaller than the type I importer cavity, TMDs of canonical ABC importers exhibit a higher although the imported substrates are even bigger in structural diversity. Moreover, as a common structural size. The transition from an inward-to-outward facing motif, the ‘coupling helices’ of canonical ABC TMD conformation is primarily mediated by a tilt of the importers are characterized by a conserved amino pore-flanking helices 3-5a. Remarkably, none of the

acid sequence (consensus EAA-X3-G-X9-I-X-LP; X, structures contains a bound substrate or nucleotide, any amino aicd) [48]. although BtuCD [46] and HI1470/71 [52] represent Crystal structures of type I ABC import systems the outward- and inward-facing TMD conformations, [36,37,49-51] revealed a similar TMD fold with a respectively, reflecting two different intermediates conserved core of six TM helices representing a of the proposed transport cycle. Furthermore, pseudo-twofold symmetry. Whereas the methionine biochemical and biophysical approaches to study transporter, MetNI, consists of only five TM helices per the reaction cycle of BtuFCD [54,55] highlighted MetI monomer [51], the N-terminal helix of each ModB substantial mechanistic differences between type I and monomer within the homodimeric molybdate/tungstate II ABC importers. While in type I systems, liganded transporter, ModBC [49], as well as of the MalG subunit SBP displays the highest binding affinity to the ATP- in the heterodimeric maltose importer [36,37] crosses bound state of the transporter (see above), the vitamin

over to interact with the opposite TMD. However, an B12 binding protein, BtuF, was shown to interact with extensive TMD-NBD domain swapping as observed in highest affinity with the substrate- and nucleotide- ABC exporters is absent. A large solvent-filled cavity free BtuCD complex. In contrast, the presence of ATP

is formed between the two TMDs of each structure, and vitamin B12 destabilized the complex and resulted displaying the substrate translocation pathway across in the dissociation of the SBP from the transporter. the cell membrane. Yet, a distinct substrate-binding Furthermore, upon ATP-binding, the NBD dimer closes pocket has so far been identified only in MalF, comprising but leads to an inward-faced TMD conformation [55]. ten residues which interact with the bound maltose [37]. Thus, the ‘alternate access’ model of translocation Based on these structures and on biochemical and within type I canonical ABC importers (Figure 4B) is biophysical evidence mainly obtained with the maltose comparable with the transport cycle proposed for ABC transporter (see [40] and further references therein), exporters, whereas the determined deviations of the the ‘alternate access’ model for type I ABC importers cycle in type II ABC import systems suggest that there was proposed (Figure 4B). Accordingly, in the absence might be a mechanistic diversity within this large protein of substrate, the transporter resides in the inward- superfamily.

792 A. Licht, E. Schneider

Figure 4. Simplified models of the translocation mechanism of ABC transporters. A. ABC exporters. In the resting state (I), the transporter is in an inward-facing conformation with a high-affinity substrate-binding site exposed to the cytoplasmic side and the inner leaflet of the membrane. Binding of substrate might occur at this stage independent of nucleotides as observed in the structure of mouse MDR1 [10] (II). Subsequent binding of ATP to the NBDs converts the transporter to an outward-facing conformation allowing extrusion of the substrate (III). ATP-hydrolysis and subsequent release of ADP restore the inward-facing conformation, thus completing the transport cycle. Structures of P-glycoprotein in the resting state and with bound substrate (black, space fill representation) which are supporting states I and II are indicated. The structure of Sav1866 complexed with the ATP analog AMP-PNP (red, space fill representation) is likely to represent the pre-hydrolysis state following release of substrate. Please note that an alternative model was proposed according to which both ATP molecules are hydrolysed sequentially (see [14] for a detailed discussion). B. Canonical ABC importers. In the resting state (I), SBP and NBDs (with bound ATP) are in their open (semi-open) conformations while the TMDs expose the substrate-binding site within the pore to the cytoplasm (inward-facing). Upon substrate binding, the SBP is shifted to the closed state (II), thereby triggering a conformational change that results in closing of the NBD dimer, opening of the TMDs to the extracellular side (outward- facing) and release of the substrate into the pore (III). ATP hydrolysis enables the TMDs to revert to the inward-facing conformation, thereby completing translocation of the substrate to the cytoplasm. Subsequent dissociation of phosphate and ADP and reloading of the NBDs with ATP restore the resting state. The model is based on known structures of the maltose transporter of E. coli, consisting of maltose binding protein, MalE (orange), the TMDs MalF (green) and MalG (light green) and a homodimer of the NBD, MalK (purple) (PDB codes are in parenthesis). In the structure representing stage III, maltose (in black, space fill representation) is bound to MalF and two ATP molecules (in red, space fill representation) are sandwiched between both MalK monomers. See text for further details.

4.2. ECF transporters protein (T component) and a transmembrane substrate- ECF (‘energy coupling factor’) transporters mediate capture protein (S component) in a stoichiometry that the uptake of mostly micronutrients (vitamins, their is still a matter of debate [56,57] but lack an SBP (for precursors and certain transition metals) in bacteria extensive review, see [8]). Based on the utilization of and archaea (see Table 2). They consist of pairs of an AAT module dedicated to a cognate S component NBDs (A components), a conserved transmembrane or shared by several S components with different

793 ATP binding cassette systems: structures, mechanisms, and functions

substrate specificities, two groups are distinguished. extracytoplasmic L1 loop capping the binding side In the widespread group I ECF transporters, all (see Figure 1). Thereby, the interactions of riboflavin components are encoded in the same operon, with the with RibU are extensive, , which is consistent with the biotin transporter BioMNY of Rhodobacter capsulatus determined high affinity dissociation constant of 0.6 nM being the best-characterized member. Interestingly, the for riboflavin [59]. solitary S component BioY has been shown to operate The least-characterized proteins of ECF transporters as an importer at high substrate concentrations (>1 nM) are the T components. Like the TMDs of canonical ABC that is converted into a high-affinity transporter (pM importers, they differ in the number and topology of range) upon association with the energizing module, TM helices and exhibit two highly conserved 3-amino- BioMN [58]. The majority of ECF transporters belong to acid signatures (consensus Ala-Arg-Gly) located at the group II, mainly found in Firmicutes and archaea, which C-terminal part corresponding to the EAA motif of TMDs comprise an AA’T module that is shared by several S from canonical importers. Mutational analysis suggested components which are encoded by genes scattered a role for intramolecular signaling and complex stability over the genome [8]. [60]. To use the energy of ATP for substrate translocation Recently, the first crystal structure of an either the S component has to interact with one or both S-component, RibU of the group II riboflavin transporter of the cytoplasmic NBDs and/or its association with of Staphylococcus aureus, was solved to 3.6 Å the T components permits the signaling via coupling of resolution. The structure revealed six transmembrane the latter with the NBDs. However, a candidate motif segments and shows a riboflavin molecule bound to was absent in the RibU structure, leaving the signaling conserved residues of TM helices 4-6 and the extended pathway currently unclear.

Transporter Transported substrate(s) Cellular function Suggested reading

Bacteria/Archaea Canonical

MalEFGK maltose, maltodextrins Nutrient supply [40]

HisJQMP positively charged amino acids Nutrient supply [41,42]

OppABCDF oligopeptides Nutrient supply, signal transduction [82]

FhuDBC Fe3+-hydroxamate (iron storage molecule) iron supply [83]

BtuFCD vitamin B12 micronutrient supply [84] LsrABCD AI-2 (autoinducer) [85]

OpuAA-OpuAB glycine-betaine (compatible solute) osmoregulation [45] ECF

BioYNM biotin micronutrient supply [8]

RibU riboflavin (vitamin 2B , precursor of FAD, FNM) micronutrient supply [8]

ThiT thiamine (precursor of vitamin B1) micronutrient supply [8] CbiMNQO Co2+ (metal cofactor of enzymes) micronutrient supply [8] Eukarya* Protozoa (Toxoplasma gondii)

ABCG5(TgABCG107) cholesterol lipid transport [62] Yeast

Aus1, Pdr11 cholesterols lipid transport [61] Plants

AtABCB14 malate modulation of stomatal movement [64]

(AtPDR12)/ABCG40 abscisic acid involvement in drought tolerance [63]

Table 2. Examples of ABC importers.

*Please note that the existence of ABC importers in eukaryotes is not yet generally recognized. See text (4.3) for further details.

794 A. Licht, E. Schneider

4.3. ABC importers in eukaryotes (reviewed in [66] and further references therein). Thus, In general, eukaryotic ABC transporters were considered how such a reversible switch from importer to exporter solely as efflux systems mediating the extrusion of activity of the very same transporter might be achieved substrates from the cytoplasm to extracytoplasmic mechanistically remains to be elucidated. compartments. However, several recent studies have The above described examples of eukaryotic import suggested the existence of ABC import systems in plants, systems are clearly homologs of eukaryotic exporters yeast and the protozoan parasite Toxoplasma gondii and thus, do not resemble canonical prokaryotic import (Table 2). Since the evidence as summarized below systems. However, within the genomes of various plant is based on in vivo data, general recognition of ABC lineages homologs of prokaryotic ECF transporter importers operating in eukaryotic cells will certainly have T components are encoded, whereas homologs of to await biochemical characterization of purified proteins. prokaryotic S components with known or predicted The first evidence came from the ABCG1-like lipid substrate specificity have not been identified to date. transporters Aus1p and Pdr11p of Saccharomyces These findings gave rise to the speculation that ECF cerevisiae which were shown to be involved in sterol transporters may exist in plants (for a more detailed uptake under anaerobiosis, when sterol biosynthesis discussion see [8]). is interfered [61]. Recently, another member of the ABCG subfamily, the ABCG5 homolog of T. gondii was implicated in lipid import as well, leading to an 5. ABC proteins not involved in accumulation of cholesterol when overexpressed in a transport mammalian cell line (COS, SV40 transformed simian kidney CV-1) [62]. Furthermore, ABCG40 of A. thaliana Besides ABC transporter proteins, cytosolic ABC mediates the uptake of the phytohormone abscisic proteins (class 2) exist, which form dimers of two acid (ABA) in guard cells, necessary for the drought tandemly repeated NBDs without a transporter function, stress response. While plant cells carrying a mutation thus leading to designation ‘ABC systems’ for the in the atabcg40 gene showed decreased ABA import, superfamily rather than ‘ABC transporters’. The lack of recombinant synthesis of the gene product in yeast TMDs indicates that substrate specificity is likely to be and BY2 (non-green tobacco callus) cells resulted in a determined by NBDs and associated domains (Table 3). raised ABA influx [63]. The DNA repair protein Rad50 of Pyrococcus Eukaryotic ABC importers have also been identified furiosus was the first structurally characterized NBD in the ABCB subfamily of plants. The ABCB14 protein demonstrated to dimerize in a head-to-tail arrangement of A. thaliana mediates uptake of malate from the upon binding of two ATP molecules. In complex with apoplast into guard cells (see ‘glossary’ for definition), Mre11 and NBS1, Rad50 mediates the homologous a process involved in the CO2-responsive regulation of recombination and joining of DNA ends via binding of closure of stomata. Recombinant production of ABCB14 DNA (reviewed in [67]). This structural arrangement was conferred malate-uptake activity on E. coli cells also observed in the crystal structure of another DNA lacking the endogenous dicarboxylate transporter and repair protein, UvrA from Bacillus stearothermophilus enhanced malate uptake of HeLa cells [64]. The ABCB1 [68]. Bacterial genomes encode various UvrA transporter (CjMDR1) of the medicinal plant Coptis paralogues, of which the DNA-binding ABC protein japonica functions as a plasma membrane berberine DrrC confers resistance to the antibiotics daunorubicin (a benzylisoquinoline alkaloid with antibacterial and and doxorubicin in the producing strain Streptomyces antimalarial activity) importer, from xylem vessels peucetius. The drrC was up-regulated into rhizome cells. This uptake activity was confirmed in the presence of daunorubicin, a direct evidence for upon production in Xenopus oozytes [65]. Finally, the

CjMDR1 homolog ABCB4 transporter of A. thaliana was Protein Cellular function Suggested demonstrated to mediate uptake of the growth hormone reading indole-3-acetic acid (auxin)- when heterologously Rad50 DNA double strand break repair [ ] expressed in HeLa cells. Surprisingly, heterologous 67 production in yeast resulted in an importer activity MutS DNA mismatch repair [86] exclusively under low auxin concentrations, whereas eEF3 elongation factor in translation [71] an increase of the intracellular auxin level caused a Uup DNA binding protein/Transposon excision [87] conversion to AtABCB4 export activity. Nevertheless, ABCE1 translation initiation, ribosome biogenesis [12] reversible auxin uptake activity of AtABCB4 has Table 3. Examples of non-transporting ABC proteins. not been observed in plant cell expression systems

795 ATP binding cassette systems: structures, mechanisms, and functions

the coupling of drug release from DNA to ATP hydrolysis 6. Concluding remarks and (see [1] for details). Yet another group of bacterial ABC systems, such perspectives as MsrA from Staphylococcus epidermidis contribute In this Mini-Review, we have addressed pro- and to the phenotype of MLS (macrolide, lincosamide eukaryotic members of the ATP-binding cassette and streptogramin) resistance. The distinct superfamily of proteins and summarized recent mechanism remains unknown. Reynolds and colleagues structural and biochemical observations that shed light [69] discussed both, an inhibitory effect on ribosome on our understanding of the mechanism(s) by which function via a direct association with the 50S subunit ABC transporters exert their functions. The available and an active efflux of MLS antibiotics by recruiting yet crystal structures of prokaryotic systems are mostly of unknown TMDs for translocation. high resolution and thus, provide reliable support for Next to the bacterial ABC systems, class II comprises the ‘alternate access’ model of transport. Nonetheless, eukaryotic members of the ABCE and ABCF subfamilies the observed differences of receptor-transporter as well. The NBDs of both families are highly conserved interactions in type I and type II importers require among eukaryotes and archaea, suggesting a fundamental additional experimental efforts for further elucidation function in the cell. For the human ABCE1, the binding of individual steps, e.g. by applying spectroscopic to RNase L has been reported, thereby modulating the approaches to transport complexes embedded in a stability of mitochondrial mRNAs, whereas the yeast lipid environment. In contrast, the resolution of the homologue Rli1p interacts with ribosomal 40S subunits only known structure of a eukaryotic system, the and the eukaryotic translation initiation factors elF-3, elF-5, P-glycoprotein, is of insufficient quality to convincingly elF-2 (reviewed in [1]). Plant Rli1p homologues have answer important questions concerning the chemical been demonstrated to participate in suppression of RNA nature of drug binding sites and conformational silencing [70]. The crystal structure of complete ABCE1 states of the TMDs. Thus, a major challenge of the from the archaeon Pyrococcus abysii revealed, besides upcoming years will be to obtain more high resolution a typical NBD dimer, a unique Fe-S-cluster domain by X-ray structures of mammalian ABC transporters with which the activity of the protein might be linked to a redox bound substrates and from different intermediate process [12]. states of the transport cycle. Such structures, together Members of the human and yeast ABCF subfamily with biochemical, biophysical and computational contribute to translational control, especially to the approaches will hopefully set the stage for developing ribosome assembly, via elF-2α kinase activation as therapies for patients suffering from diseases caused in case of the yeast GCN20 NBD, while the yeast by defective ABC transporters or for selectively elongation factor EF-3 is responsible for the proper inactivating ABC drug exporters in the course of loading of the aminoacyl tRNAs on the ribosome [1,71]. anticancer chemotherapy. In conclusion, ABC non-transporters assume mostly regulatory functions in fundamental cellular processes as DNA repair or ribosomal translation. At least some Acknowledgements bacterial members contribute to drug resistance, which could be regarded as a novel self-resistance Work from the authors’ laboratory was supported by the mechanism in antibiotic-producing organisms, enabling Deutsche Forschungsgemeinschaft (SFB 449, TP B14; the establishment in ecological niches. SCHN 274/9-3; 14-1; 15-1).

796 A. Licht, E. Schneider

Glossary Major histocompatibility complex (MHC): large gene family of MHC molecules found in most vertebrates, Adrenoleukodystrophy: rare inherited metabolic which display peptide fragments of processed proteins disorder most often caused by a defect in the ABCD1 on the cell surface (‘antigen presentation’). MHC class gene of the ALDP exporter affecting the myelin function I molecules, found on all nucleated cells, present of the central and peripheral nervous system which antigens to T cells, whereas MHC class II molecules are leads to a progressive brain damage. found on different antigen-presenting cells specialized Anthracyclines: class of antibiotic chemotherapeutic in pathogen uptake. agents originally derived from the soil bacterium genus MLS antibiotics (macrolide, lincosamide, Streptomyces. They are widely used against leukemia streptogramin): group of structurally diverse antibiotics due to attacking cancer cells by DNA intercalation, that act as translation inhibitors on the bacterial 50S inhibiting replication and leading to cell death. The two ribosome. Three different resistance mechanisms have most common used derivatives are daunorubicin and been described; target modification by rRNA methylases doxorubicin. (erm), chemically modification and thus inactivation of Apoplast: free diffusional space outside the the antibiotics (ereA/B) or presence of multicomponent plasma membrane in any plant tissue, comprising the macrolide efflux pumps (msrA/B or mefA/E). continuum of cell walls of adjacent cells as well as the Sitosterolemia: rare inherited lipid metabolic extracellular spaces. The apoplast is important for inter disorder characterized by increased amounts of plant cellular communication. sterols (sitosterol, stigmasterol, campesterol, and their Cystic fibrosis: common autosomal recessive 5-alpha derivatives) in blood and various tissues leading genetic disease caused by a mutation (most often to hypercholesterolemia as a result of mutated sterolin-1 ΔF508) of the CFTR (Cystic Fibrosis Transmembrane and sterolin-2 ABC exporter. Regulator). The dysfunction of the CFTR chloride ion Stargardt’s macular degeneration: also known as channel results in a thickened exocrine gland secret Stargardt macular dystrophy, the most common form causing progressive disability and often early death. of inherited juvenile macular degeneration causing a Diabetes mellitus: one of the most common progressive loss of central vision. Due to a dysfunctional metabolic disorders with an abnormal high level of blood ABCA4 transporter, yellow fatty material accumulates in glucose due to a destruction of the insulin producing the retina leading to a degeneration of photoreceptor beta cells in the pancreas (type 1, insulin dependent) or cells. a lack of sensitivity to insulin by mostly fat and muscle Tangier disease: rare inherited metabolic disorder cells (type 2, non-insulin dependent). characterized by a reduced amount of high density Dilated cardiomyopathy: the most common lipoprotein (HDL) due to a defective ABCA1 transporter form of non-ischemic cardiomyopathy characterized preventing an effective cholesterol cell export. by a weakend and enlarged heart often caused by a Vinca alkaloids: anti-mitotic and anti-microtubule myocardium damage probably due to exposure to toxic, agents preventing tubulin formation and thus affecting metabolic or infectious agents. cell division. Derived from the Madagascar periwinkle Guard cells: specialized cells on the undersurface plant or produced synthetically, these agents are of leaves surrounding stomatas for controlling gas mostly used in cancer treatment preventing cancer exchange and water loss. cell growth. The four major vinca alkaloids in clinical HeLa cells: first continuous cancer cell line derived use are vinblastine, vinorelbine, vincristine, and from a patient by the name of Henrietta Lacks in 1951. vindesine.

797 ATP binding cassette systems: structures, mechanisms, and functions

References

[1] Davidson, A.L., Dassa, E., Orelle, C., Chen, J., [15] Yazaki K., Shitan N., Sugiyama A., Takanashi T., Structure, function, and evolution of bacterial ATP- Cell and molecular biology of ATP-binding cassette binding cassette systems, Microbiol. Mol. Biol. proteins in plants, In: Jeon K.W., (Ed.), International Rev., 2008, 72, 317-364 review of cell and molecular biology, Vol. 276, [2] Vasiliou V., Konstandinos Vasiliou K., Nebert D.W., Academic Press, Burlington, 2009, 263-299 Human ATP-binding cassette (ABC) transporter [16] Kerr I.D., Jones P.M., George A.M., Multidrug efflux family, Hum. Genomics, 2009, 3, 281-290 pumps: The structures of prokaryotic ATP-binding [3] Moussatova A., Kandt C., O’Mara M.L., Tieleman cassette transporter efflux pumps and implications D.P., ATP binding cassette transporters in for our understanding of eukaryotic P-glycoproteins Escherichia coli, Biochim. Biophys. Acta, 2008, and homologues, FEBS J., 2010, 277, 550-563 1778, 1757-1771 [17] Holland I.B., Cole S.P.C., Kuchler K., Higgins [4] Rea P.A., Plant ATP-binding cassette transporters, C.F., (Eds.) ,ABC proteins: From bacteria to man, Annu. Rev. Plant Biol., 2007, 58, 347–375 Elsevier, Amsterdam, 2003 [5] Schneider E., Hunke S., ATP-binding-cassette [18] Sperandeo P., Dehò G., Polissi A., The (ABC) transport systems: Functional and structural lipopolysaccharide transport system of Gram- aspects of the ATP-hydrolyzing subunits/domains, negative bacteria, Biochim. Biophys. Acta, 2009, FEMS Microbiol. Rev., 1998, 22, 1-20 1791, 594-602 [6] Oswald C., Holland I.B., Schmitt L., The motor [19] Schölz C., Tampé R., The peptide-loading complex- domains of ABC-transporters. What can structures antigen translocation and MHC class I loading, Biol. tell us?, Naunyn-Schmiedeberg’s Arch. Pharmacol., Chem., 2009, 390, 783-794 2006, 372, 385-399 [20] Szakács G., Paterson J.K., Ludwig J.A., Booth- [7] Jones P.M., O’Mara M.L., George A.M., ABC Genthe C., Gottesman M.M., Targeting multidrug transporters: a riddle wrapped in a mystery resistance in cancer, Nat. Rev. Drug Discov., 2006, inside an enigma, Trends Biochem. Sci., 2009, 5, 219-234 34, 520-531 [21] Ernst R., Kueppers P., Stindt J., Kuchler K., Schmitt [8] Eitinger T., Rodionov D.A., Grote M., Schneider L., Multidrug efflux pumps: substrate selection in E., Canonical and ECF-type ATP binding cassette ATP-binding cassette multidrug efflux pumps-first importers in prokaryotes: diversity in modular come, first served?, FEBS J., 2010, 277, 540-549 organisation and cellular functions, FEMS Microbiol. [22] Ward A., Reyes C.L., Yu J., Roth C.B., Chang G., Rev., 2011, 35, 3-67 Flexibility in the ABC transporter MsbA: Alternating [9] Rees D.C., Johnson E., Lewinson O., ABC access with a twist, Proc. Natl. Acad. Sci. U S A., transporters: the power to change, Nature Rev. 2007, 104, 19005-19010 Mol. Cell Biol., 2009, 10, 218-227 [23] Dawson R. J. P., Locher K. P., Structure of [10] Aller S.G., Yu J., Ward A., Weng Y., Chittaboina S., the multidrug ABC transporter Sav1866 from Zhuo R., et al., Structure of P-glycoprotein reveals Staphylococcus aureus in complex with AMP-PNP, a molecular basis for poly-specific drug binding, FEBS Lett., 2007, 581, 935-938 Science, 2009, 323, 1718-1722 [24] Oancea G., O’Mara M.L., Bennett W.F., Tieleman [11] Zhang P., Wang J., Shi Y., Structure and mechanism D.P., Abele R., Tampé R., Structural arrangement of the S component of a bacterial ECF transporter, of the transmission interface in the antigen ABC Nature, 2010, 468, 717-720 transport complex TAP, Proc. Natl. Acad. Sci. USA, [12] Karcher A., Schele A., Hopfner K.P., X-ray 2009, 106, 5551-5556 Structure of the complete ABC enzyme ABCE1 [25] Dawson R.J., Locher K.P., Structure of a bacterial from Pyrococcus abyssi, J. Biol. Chem., 2008, 283, multidrug ABC transporter, Nature, 2006, 443, 7962-7971 180-185 [13] Borst P., Elferink R.O., Mammalian ABC transporter [26] Gupta R.P., Kueppers P., Schmitt L., Ernst R., The in health and disease, Annu. Rev. Biochem., 2002, multidrug transporter Pdr5: a molecular diode?, 71, 537-592 Biol. Chem., 2011, 392, 53-60 [14] Seeger M.A., van Veen H.W., Molecular basis of [27] Locher K.P., Structure and mechanism of ATP- multidrug transport by ABC transporters, Biochim. binding cassette transporters, Phil. Trans. R. Soc. Biophys. Acta, 2009, 1794, 725-737 B, 2009, 364, 239-245

798 A. Licht, E. Schneider

[28] Federici L., Woebking B., Velamakanni S., Shilling [40] Bordignon E., Grote M., Schneider E., The maltose R.A., Luisi B., van Veen H.W., New structure model ABC transporter in the 21st century – towards a for the ATP-binding cassette multidrug transporter structural-dynamic perspective on its mode of LmrA, Biochem. Pharmacol., 2007, 74, 672-678 action, Mol. Microbiol., 2010, 77, 1354-1366 [29] Velamakanni S., Yao Y., Gutmann D.A.P., van Veen [41] Schneider E., Import of solutes by ABC transporters- H.W., Multidrug Transport by the ABC Transporter the maltose and other systems, In: Holland E.B., Cole Sav1866 from Staphylococcus aureus, 2008, S., Kuchler K., Higgins C., (Eds.), ABC proteins: From Biochemistry 47, 9300-9308 bacteria to man, Elsevier, Amsterdam, 2003, 157-185 [30] Hwang T.C., Sheppard D.N., Gating of the CFTR [42] Ames G.F., Nikaido K., Wang I.X., Liu P.Q., Liu Cl- channel by ATP-driven nucleotide-binding C.E., Hu C., Purification and characterization of the domain dimerization, J. Physiol., 2009, 587, membrane-bound complex of an ABC transporter, 2151-2161 the histidine permease, J. Bioenerg. Biomembr., [31] Bryan J., Muñoz A., Zhang X., Düfer M., Drews G., 2001, 33, 79-92 Krippeit-Drews P., et al., ABCC8 and ABCC9: ABC [43] Hung L.-W.,Wang I.X., Nikaido K., Liu P.-Q., Ames transporters that regulate K+ channels, Pflugers G.F.-L., Kim S.-H., Crystal structure of the ATP- Arch., 2007, 453, 703-718 binding subunit of an ABC transporter, Nature, [32] Berntsson R.P., Smits S.H., Schmitt L., Slotboom 1998, 396, 703-707 D.J., Poolman B., A structural classification of [44] Wood J.M., Osmosensing by bacteria: signals and substrate-binding proteins, FEBS Lett., 2010, 584, membrane-based sensors, Microbiol. Mol. Biol. 2606-2617 Rev., 1999, 63, 230-262 [33] Shilton B.H., The dynamics of the MBP-MalFGK(2) [45] Patzlaff J.S., van der Heide T., Poolman B., The interaction: a prototype for binding protein ATP/substrate stoichiometry of the ATP-binding dependent ABC-transporter systems, Biochim. cassette (ABC) transporter OpuA, J. Biol. Chem., Biophys. Acta, 2008, 1778, 1772-1780 2003, 278, 29546-29551 [34] Schneider E., Eckey V., Weidlich D., Wiesemann [46] Locher K.P., Lee A.T., Rees D.C., The E. coli N., Vahedi-Faridi A., Thaben P., et al., Receptor- BtuCD structure: a framework for ABC transporter transporter interactions of canonical ATP-binding architecture and mechanism, Science, 2002, 296, cassette import systems in prokaryotes, Eur. J. Cell 1091-1098 Biol., 2011, (in press) doi:10.1016/j.ejcb.2011.02.008 [47] Locher K., Structure and mechanism of ATP-binding [35] Falke J.J., Hazelbauer G.L., Transmembrane cassette transporters, Phil. Trans. R. Soc. B, 2009, signaling in bacterial chemoreceptors, Trends 364, 239-245 Biochem. Sci., 2001, 26, 257-265 [48] Saurin W., Köster W., Dassa E., Bacterial binding [36] Khare D., Oldham M., Orelle C., Davidson A.L., protein dependent permeases: characterization of Chen J., Alternating access in maltose transporter distinctive signatures for functionally related integral mediated by rigid-body rotations, Mol. Cell, 2009, cytoplasmic membrane proteins, Mol. Microbiol., 33, 528-536 1994, 12, 993-1004 [37] Oldham M. L., Khare D., Quiocho F.A., Davidson [49] Hollenstein K., Frei D.C., Locher K.P., Structure A.L., Chen J., Crystal structure of a catalytic of an ABC transporter in complex with its binding intermediate of the maltose transporter, Nature, protein, Nature, 2007, 446, 213-216 2007, 450, 515-522 [50] Gerber S., Comellas-Bigler M., Goetz B. A., [38] Daus M.L., Grote M., Schneider E., The Locher K. P., Structural basis of trans-inhibition in MalF-P2 loop of the ATP-binding cassette a molybdate/tungstate ABC transporter, Science, (ABC) transporter MalFGK2 from Escherichia 2008, 321, 246-250 coli / Salmonella enterica serovar Typhimurium [51] Kadaba N. S., Kaiser J. T., Johnson E., Lee A., interacts with maltose binding protein (MalE) Rees D. C., The high-affinity E. coli methionine throughout the catalytic cycle, J. Bacteriol., 2009, ABC transporter: structure and allosteric regulation, 191, 754-761 Science, 2008, 321, 250-253 [39] Grote M., Polyhach Y., Jeschke G., Steinhoff H.- [52] Pinkett H. W., Lee A. T., Lum P., Locher K. P., Rees J., Schneider E., Bordignon E., Transmembrane D. C., An inward-facing conformation of a putative signaling in the maltose ABC transporter MalFGK2-E. metal-chelate type ABC transporter, Science, 2007, Periplasmic MalFP2 loop communicates substrate 315, 373-377 availability to the ATP-bound MalK dimer, J. Biol. [53] Hvorup R.N., Goetz B.A., Niederer M., Hollenstein Chem., 2009, 284, 17521-17526 K., Perozo E., Locher K.P., Asymmetry in the

799 ATP binding cassette systems: structures, mechanisms, and functions

structure of the ABC transporter-binding protein [65] Shitan N., Bazin I., Dan K., Obata K., Kigawa K., complex BtuCD-BtuF, Science, 2007, 317, 1387- Ueda K., et al., Involvement of CjMDR1, a plant 1390 multidrug-resistance-type ATP-binding cassette [54] Goetz B.A., Perozo E., Locher K.P., Distinct gate protein, in alkaloid transport in Coptis japonica, conformations of the ABC transporter BtuCD Proc. Natl. Acad. Sci. USA, 2003, 100, 751-756 revealed by electron spin resonance spectroscopy [66] Zazímalová E., Murphy A.S., Yang H., Hoyerová K., and chemical crosslinking, FEBS Lett., 2009, 583, Hosek P., Auxin transporters-why so many?, Cold 266-270 Spring Harb. Perspect. Biol., 2010, 2, a001552 [55] Lewinson O., Lee A.T., Locher K.P., Rees D.C., A [67] Hopfner K.P., Tainer J.A., Rad50/SMC proteins and distinct mechanism for the ABC transporter BtuCD– ABC transporters: unifying concepts from high- BtuF revealed by the dynamics of complex formation, resolution structures, Curr. Opin. Struct. Biol., 2003, Nature Struct. Mol. Biol., 2010, 17, 332-338 13, 249-255 [56] Finkenwirth F., Neubauer O., Gunzenhäuser [68] Pakotiprapha D., Inuzuka Y., Bowman B.R., Moolenaar J., Schoknecht J., Scolari S., Stöckl M., et al., G.F., Goosen N., Jeruzalmi et al., Crystal structure of Subunit composition of an energy-coupling-factor- Bacillus stearothermophilus UvrA provides insight into type biotin transporter analysed in living bacteria, ATP-modulated dimerization, UvrB interaction, and Biochem. J., 2010, 431, 373-380 DNA binding, Mol. Cell, 2008, 29, 122-133 [57] ter Beek J., Duurkens R.H., Erkens G.B., Slotboom [69] Reynolds E., Ross J.I., Cove J.H., Msr(A) and D.J., Quaternary structure and functional unit of related macrolide/ streptogramin resistance energy coupling factor (ECF)-type transporters, J. determinants: incomplete transporters?, Int. J. Biol. Chem., 2011, 286, 5471-5475 Antimicrob. Agents, 2003, 22, 228-236 [58] Hebbeln P., Rodionov D.A., Alfandega A., Eitinger T., [70] Braz A.S., Finnegan J., Waterhouse P., Margis R., Biotin uptake in prokaryotes by solute transporters A plant orthologue of RNase L inhibitor (RLI) is with an optional ATP-binding cassette-containing induced in plants showing RNA interference, J. Mol. module, Proc. Natl. Acad. Sci, U S A, 2007, 104, Evol., 2004, 59, 20-30 2909-2914 [71] Andersen C.B.F., Becker T., Blau M., Anand M., [59] Zhang P., Wang J., Shi Y., Structure and mechanism Halic M., Balar B., et al., Structure of eEF3 and the of the S component of a bacterial ECF transporter, mechanism of transfer RNA release from the E-site, Nature, 2010, 468, 717-720 Nature, 2006, 443, 663-668 [60] Neubauer O., Alfandega A., Schoknecht J., [72] Zaitseva J., Jenewein S., Oswald C., Jumpertz T., Sternberg U., Pohlmann A., Eitinger T., Two Holland I.B., Schmitt L., A molecular understanding essential arginine residues in the T components of of the catalytic cycle of the nucleotide-binding energy-coupling factor transporters, J. Bacteriol., domain of the ABC transporter HlyB, Biochem. Soc. 2009, 191, 6482-6488 Trans., 2005, 33, 990-995 [61] Wilcox L.J., Balderes D.A., Wharton B., Tinkelenberg [73] van Veen H.W., Callaghan R., Soceneantu L., A.H., Rao, G. Sturley S.L., Transcriptional profiling Sardini A., Konings W.N., Higgins C.F., A bacterial identifies two members of the ATP-binding cassette antibiotic-resistance gene that complements the transporter superfamily required for sterol uptake in human multidrug-resistance P-glycoprotein gene, yeast, J. Biol. Chem., 2002, 277, 32466-32472 Nature, 1998, 391, 291-295 [62] Ehrenman K., Sehgal A., Lige B., Stedman T.T., [74] Zhang H., Pradhan P., Kaur P., The extreme C Joiner K.A., Coppens I., Novel roles for ATP-binding terminus of the ABC protein DrrA contains unique cassette G transporters in lipid redistribution in motifs involved in function and assembly of the DrrAB Toxoplasma, Mol. Microbiol., 2010, 76, 1232-1249 complex, 2010, J. Biol. Chem. 285, 38324-38336 [63] Kang J., Hwang J.-U., Lee M., Kim Y.-Y., Assmann [75] Tokuda H., Biogenesis of outer membranes S.M., Martinoia E., et al., PDR-type ABC transporter in Gram-negative bacteria, Biosci. Biotechnol. mediates cellular uptake of the phytohormone Biochem., 2009, 73, 465-473 abscisic acid, Proc. Natl. Acad. Sci. U S A, 2010, [76] Berkower C., Taglicht D., Michaelis S., Functional 107, 2355-2360 and physical interactions between partial molecules [64] Lee M., Choi Y., Burla B., Kim Y.Y., Jeon B., of STE6, a yeast ATP-binding cassette protein, J. Maeshima M., et al., The ABC transporter Biol. Chem., 1996, 271, 22983-22989 AtABCB14 is a malate importer and modulates [77] Zuben E., Sauna Z.E., Kim I.-W., Ambudkar S.V.,

stomatal response to CO2, Nature Cell Biol., 2008, Genomics and the mechanism of P-glycoprotein 10, 1217-1223 (ABCB1), J. Bioenerg. Biomembr., 2007, 39, 481-487

800 A. Licht, E. Schneider

[78] Berger J., Gärtner J., X-linked adrenoleukodystrophy: Transport in Bacteria, Crosa J.H., Mey A.R., clinical, biochemical and pathogenetic aspects, Payne S.M., (Eds.), ASM Press, Washington, Biochim. Biophys. Acta, 2006, 1763, 1721-1732 DC, 2004, 158-177 [79] Molday R.S., Zhong M., Quazi F., The role of the [84] Borths E.L., Poolman B., Hvorup R.N., Locher photoreceptor ABC transporter ABCA4 in lipid K.P., Rees D.C., In vitro functional characterization transport and Stargardt macular degeneration, of BtuCD-F, the Escherichia coli ABC transporter Biochim. Biophys. Acta, 2009, 1791, 573-583 for vitamin B12 uptake, Biochemistry, 2005, 44, [80] Attie A.D., ABCA1: at the nexus of cholesterol, HDL 16301-16309 and atherosclerosis, Trends Biochem. Sci., 2007, [85] Li J., Attila C., Wang L., Wood T.K., Valdes J.J., Bentley 32, 172-179 W.E., Quorum sensing in Escherichia coli is signaled [81] Gadsby D.C., Vergani P., Csanády L., The ABC by AI-2/LsrR: effects on small RNA and protein turned chloride channel whose failure architecture, J. Bacteriol., 2007, 189, 6011-6020 causes cystic fibrosis, Nature, 2006, 440, 477-483 [86] Kunkel T.A., Erie D.A., DNA mismatch repair, Annu. [82] Doeven M.K., Abele R., Tampé R., Poolman B., The Rev. Biochem., 2005, 74, 681-710 binding specificity of OppA determines the selectivity [87] Zepeda M.Y.B., Alessandri K., Murat D., El Amri of the oligopeptide ATP-binding cassette transporter, C., Dassa E., C-terminal domain of the Uup ATP- J. Biol. Chem., 2004, 279, 32301-32307 binding cassette ATPase is an essential folding [83] Braun V., Braun M., Killmann H., Ferrichrome- domain that binds to DNA, Biochim. Biophys. Acta, and citrate-mediated iron transport, In: Iron 2010, 1804, 755-761

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