Review

Understanding polyspecificity ofmultidrug ABC transporters: closingin on the gaps in ABCB1Daniel A.P. Gutmann1, Andrew Ward2, Ina L. Urbatsch3, Geoffrey Chang2 andHendrik W. van Veen1

1 Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK2 Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, CB105, La Jolla, CA 92037, USA3 Cell Biology and Biochemistry, and Center for Membrane Protein Research, Texas Tech University Health Sciences Center,

3601 4th Street, Lubbock, TX 79430, USA

Multidrug ABC transporters can transport a wide rangeof drugs from the cell. Ongoing studies of the prototypemammalian multidrug resistance ATP-binding cassettetransporter P-glycoprotein (ABCB1) have revealed manyintriguing functional and biochemical features. How-ever, a gap remains in our knowledge regarding themolecular basis of its broad specificity for structurallyunrelated ligands. Recently, the first crystal structures ofligand-free and ligand-bound ABCB1 showed ligandbinding in a cavity between its two membrane domains,and earlier observations on polyspecificity can now beinterpreted in a structural context. Comparison of thenew ABCB1 crystal structures with structures of bac-terial homologs suggests a critical role for an axialrotation of transmembrane helices for high-affinity bind-ing and low-affinity release of ligands during transmem-brane transport.

Domain organisationMultidrug ATP-binding cassette (ABC) transporters med-iate the ATP-dependent extrusion of cytotoxic agents awayfrom their intracellular targets [1]. They are pharmaco-logically important proteins in humans that participate inboth the distribution of drugs in the body and eliminationof drugs from the body, and can confer drug resistance oncancer cells [2–4]. These transporters also are expressed inplants [5] and inmicrobial pathogens associated with someof the most devastating diseases in the world; in thiscapacity they can impair antimicrobial chemotherapy[6–8]. Multidrug ABC transporters belong to the ABCsuperfamily that contains 48 representatives in the humangenome alone. In this superfamily, ABC exporters can bedistinguished from ABC importers by the directionality oftransport and distinct structural arrangements of themembrane domains (MDs) [9]. All ABC transporters con-tain two nucleotide-binding domains (NBDs), each carry-ing the namesake ABC motif, and two MDs, usually eachcontaining six transmembrane helices (TMHs) in expor-ters. In Bacteria and Archaea, ABC exporters are typicallyexpressed as half-transporters, with one NBD and one MDon a single polypeptide chain. Two chains then assemble

Corresponding author: van Veen, H.W. (hwv20@cam.ac.uk).

36 0968-0004/$ – see front matter � 2009 Elsevie

into a functional homo- or heterodimer. In Eukarya, how-ever, ABC exporters are often expressed as a single poly-peptide chain containing the four domains. The humanmultidrug resistance P-glycoprotein ABCB1, which wasfirst described by Danø [10] and Juliano and Ling [11],and subsequently cloned as a full-length cDNA by Uedaand colleagues [12], is a typical example of this architec-ture.

In general, the dimeric NBDs in multidrug ABC trans-porters act in concert to hydrolyze ATP and provide the freeenergy to drive directional transport against transmem-brane concentration gradients for hydrophilic substratesand against the lipid–water partition coefficient for hydro-phobic substrates. Several models have been proposed toexplain the energetic coupling between the NBDs and thetransport by the MDs (reviewed in Ref. [13]) but they arenot discussed here. Instead, we aim to provide the readerwith an up-to-date view of drug–multidrug ABC transpor-ter interactions. We address, in particular, the structuralfeatures and mechanisms that allow the MDs of ABCB1and bacterial homologs to bind and transport toxic ions anddrugs (referred to as ligands). By comparing the recentlypublished crystal structure of ligand-bound ABCB1 withthe available crystal structures of bacterial homologs inpost hydrolysis states, we suggest a role of helix rotation inligand binding and release on opposite sides of the mem-brane.

Drug binding and transportOur early knowledge about the ligand specificity of multi-drug ABC transporters comes, in large part, from cellbiological and biochemical experimentation. One recurrenttheme that emerged from these studies is that hydrophobicligandsmight interact with binding sites in ABCB1 that liewithin the membrane. For example, the potency of inhibi-tors to modify ABCB1-mediated anthracycline transport isdirectly proportional to their ability to partition in thephospholipid bilayer [14]. Non-fluorescent acetoxymethylprecursors of calcein (calcein-AM) and 20,70-bis-(2-carbox-yethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) areextruded by ABCB1 and other systems before these pre-cursors are converted into fluorescent calcein and BCECFprobes by cytoplasmic non-specific esterases [15,16].

r Ltd. All rights reserved. doi:10.1016/j.tibs.2009.07.009 Available online 12 October 2009

Review Trends in Biochemical Sciences Vol.35 No.1

The transport of the hydrophobic fluorescent dye 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene(TMA-DPH) occurs at a rate dependent on its concen-tration in the inner leaflet of the membrane [16]. Fluor-escence energy transfer experiments on ABCB1 positioneda binding site for the dye Hoechst 33342 in the innermembrane leaflet [17]. In another setting, the ABCB1homolog HlyB from Escherichia coli interacts with thesignal sequence of a-hemolysin that forms an amphiphilichelix and binds to the cytoplasmic leaflet of the plasmamembrane [18]. These examples support the notion thatamphiphilic and hydrophobic ligands are ‘intercepted’while they reside at the membrane, raising the suggestionthat ABCB1 acts by a hydrophobic vacuum cleaner mech-anism [19,20]. The physiological relevance of this mech-anism for multidrug ABC transporters is related to theobserved tendency of many antimicrobial and anticancerchemotherapeutic agents to accumulate in the water–lipidinterfaces of the membrane [21], and for intracellulardrugs in the interface associated with the inner membraneleaflet [16].

Various studies have attempted to locate the drug-bind-ing sites and key residues responsible for the interactionwith ligands. In addition to ABCB1 [22], the bacterialhomologs LmrA [23–25] from Lactococcus lactis and MsbA[26–30] from E. coli are among the best-studied multidrugABC transporters. The range of ligands that can be trans-ported by these ABC systems overlaps, and LmrA canfunctionally substitute for ABCB1 in human lung fibro-blast cells [31]. The MDs in ABCB1 and homodimericLmrA share the common 6 + 6 helix arrangement [32]that enables binding of ligands in the absence of the NBDs[24,33]. Studies of ligand–ligand interactions on ABCB1revealed that some ligands interact with the transporter assingle molecules, whereas others interact as pairs [34].ABCB1 contains distinct sites for transport of rhodamine123 (R-site) and Hoechst 33342 (H-site) in addition to amodulatory site for prazosin and progesterone [35,36]. Theinteraction of ligand at one of the transport competent sitesenhances the ligand interaction at the other site [35].LmrA is equally capable of binding and transporting twodrug molecules in a positive cooperative manner [23],further underlining the mechanistic similarities betweenmammalian and bacterial multidrug ABC transporters.Equilibrium binding measurements on ABCB1 providedevidence for three sites for transported ligands (vinblas-tine, pacitaxel, and Hoechst 33342), which can interactwith ligand in the absence of exogenously added nucleo-tide, in addition to a modulatory site for nicardipine/GF120918 [37]. As a network of interactions between thesekinetically distinguishable drug-binding sites exists, thepossibility was raised that these sites are present on acommon binding surface.

Cross-linking experiments using photoreactive druganalogs followed by peptide mapping [38] provided a firstglance at the locations of ligand binding in ABCB1. Theseinsights were expanded by extensive cysteine scanningmutagenesis in which single mutants were tested for theirability to react with thiol-reactive substrates includingdibromobimane [39], methanethiosulfonate-verapamil(MTS-verapamil) [40], and MTS-rhodamine B [41]. The

non thiol-containing verapamil and rhodamine substrateswere able to protect cysteines against labeling, therebyindicating that their MTS containing analogs boundABCB1 at overlapping sites. The resulting data showedthat residues on TMHs 6, 9 and 12 form the R-site pocket,whereas the H-site is created by residues located on TMHs4, 6, 10, 11 and 12. A role for TMH 6 in drug binding wasfound also in MsbA, where Ser289 and Ser290 to Ala(SASA) substitutions in TMH 6 specifically inhibit theinteraction ofMsbAwith ethidium and the anticancer drugTaxol [28]. Further studies examined the solvent accessi-bility of these regions. In LmrA, the highly soluble thiol-reactive compound fluorescein maleimide could access 11out of 15 aromatic residues in the MD [42]. In similarexperiments on ABCB1, the water-soluble cysteine-reac-tive compounds sodium (2-sulfonatoethyl) methanethio-sulfonate (MTS-ES) and [2-(trimethylammonium)ethyl)]methanethiosulfonate (MTS-ET) competed successfullywith Verapamil for site-specific labeling of single cysteinemutants [43]. As all of the experiments were carried out inthe absence of ATP or non-hydrolysable nucleotide ana-logs, it was concluded that ABC multidrug transporterscontain ligand-binding chambers that are exposed to thecytoplasm and are partially hydrated.

The newly published inward-facing structures ofABCB1 [44] exhibit structural features that give furthercredence to the previously discussed observations. At3.8 A, the crystal structure for apo ABCB1 is of moderateresolution with 65.8% in most favored regions, 26.7% inadditional allowed regions, 7.5% in generously allowedregions and, most importantly at this resolution, 0% indisallowed regions (determined by ProCheck). The pos-itions of residues were also verified by composite Fo-Fcdifference maps, which are more rigorous than the usual2Fo-Fc electron-density maps. The MDs indeed form aligand-binding chamber that is exposed to the cytoplasmand contains a laterally open gap that is accessible to theinner leaflet of the membrane (Figure 1a). The V-shapedarrangement of the MDs in the membrane is also observedin the inward-facing structures of MsbA from E. coli andVibrio cholera [45] (Table 1). This conformation is sup-ported by recent pulse double electron–electron resonanceand fluorescence homo-transfer experiments using puri-fied E. coli MsbA in detergent solution as well as wheninserted in the phospholipid bilayer of liposomes [46]. Inthe outward-facing nucleotide-bound conformations of theABCB1 homologs MsbA from Salmonella typhimurium[45] and Sav1866 from Staphylococcus aureus [47,48],and our ABCB1 models based on these templates(Figure 1b), theMDs also form a lateral gap that is exposedto the outer leaflet of the bilayer and the external environ-ment. The inward-facing gap in ABCB1 (Figure 1a) andpredicted outward-facing gap (Figure 1b) are connected bythe ligand-binding surface at the center of the membrane[44] (Figure 1c). This ligand-binding surface might bepreserved in the same position in the bacterial ABCB1homologs. Residues Ser289 and Ser290 in E. coli MsbAthat contribute to ligand specificity of the transporter [28]are located at positions corresponding to those in TMH 6and 12 of inward-facing ABCB1 at the top of the bindingcavity for the co-crystallized cyclic peptide P-glycoprotein

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Figure 1. Model for ligand efflux by ABCB1. ABCB1 contains a central ligand-

binding cavity close to the leaflet–leaflet interface of the membrane. During

transport, this binding cavity is alternately exposed to the inside and the outside

surface of the membrane. (a) The inward-facing structure of ABCB1 and (b) the

putative outward-facing structure have a laterally open cleft in their MDs exposed

to the lipid phase of the bilayer. In the inward-facing conformation, this ‘gap’ could

grant ligand (green) access to the binding site directly from the inner leaflet of the

phospholipid bilayer and cytoplasm. When the transporter assumes its outward-

facing conformation, the ligand is expelled into the outer leaflet and/or aqueous

extracellular medium. The outward-facing ABCB1 model in (b) is based on MsbA

from S. typhimurium [45] and Sav1866 from S. aureus [47]. (c) Cross-section

through ABCB1 bound to two molecules of QZ59-SSS (black) reveals the central

location of the ligand-binding site in relation to the phospholipid bilayer. The

residues in ABCB1 corresponding to Ser289 and Ser290 in E. coli MsbA, which

affect ligand specificity of this bacterial transporter [28], are shown as brown

spheres to highlight their juxtaposition to the bound ligand. The surface is

rendered by hydrophobicity (orange) and hydrophilicity (blue). The model in (b)

was generated using Modeller. This figure was generated in Chimera.

Review Trends in Biochemical Sciences Vol.35 No.1

inhibitors (CPPIs; QZ59-RRR and QZ59-SSS) [44], close tothe leaflet–leaflet interface (Figure 1c). The positioning ofthe binding cavity at the center of the phospholipid bilayerlowers the activation energy for permeation of ligand

Table 1. Available crystal structures of multidrug ABC exporters

Protein Conformationa Nucleotide

1 Sav1866 Outward ADPb

2 Sav1866 Outward AMP-PNP

3 MsbA Inward –

4 MsbA Inward –

5 MsbA Outward AMP-PNP

6 MsbA Outward ADP-OV

7 MsbA Outward AMP-PNP

8 Mouse ABCB1a Inward –

9 Mouse ABCB1a Inward –

10 Mouse ABCB1a Inward –aConformation refers to the inward or outward-facing orientations of the cavity formedbAbbreviations: ADP, adenosine diphosphate; AMP-PNP, adenosine 50-(b,g-imido)tripho

valineselenazole QZ59-SSS cyclic-tris-(S)-valineselenazole.

38

through the acyl chain region, and grants alternatingaccess to the cytosolic and extracellular-facing leaflets ofthe membrane in a fashion that would not require signifi-cant movement of the ligand. Ligand binding close to theleaflet–leaflet interface also is observed for ABC importers[9] and for members of the major facilitator superfamily(MFS) [49], which represents the largest group of second-ary-activemembrane transporters. The crystal structure ofthe lactose-H+ symporter LacY from E. coli shows thelactose-binding site in a hydrophobic environment at thecenter of the membrane, consistent with the observationthat the affinity of this MFS transporter for dansylatedsugars increases with the hydrophobicity of the sugarderivatives [50]. Similarly, the ligand-binding site of thehuman MFS transporter GLUT1 (for glucose) can belabeled with lipophilic forskolin derivatives [51]. Hence,although sugars are not lipid-soluble substrates, theirbinding sites in sugar transporters can be accessible fromboth the aqueous phase and the phospholipid bilayer.EmrD, whose drug-binding site appears accessible fromthe lipid bilayer in a crystal structure [52], transportsuncouplers of oxidative phosphorylation like meta-chlorocarbonylcyanide phenylhydrazone (CCCP) directly fromthe cell membrane. The similarities in the location andaccessibility of ligand binding sites across a range oftransporters might translate into mechanistic similaritiesas recent studies highlighted a functional analogy betweensecondary-active transporters and LmrA [24,25]. Amphi-philic ligands, which insert with their hydrophobic moi-eties in the acyl chain region of one leaflet of thephospholipid bilayer and with their hydrophilic moietiesin the polar head group region of that leaflet, are likely tobe inverted during alternating access by ABCB1 by amechanism that is not resolved experimentally. Thus,the ‘ON’ and ‘OFF’ sites originally proposed by Ambudkarand colleagues [53] could reflect alternating access of thesame binding cavity to the inner and outer membraneleaflets. The possibility exists, however, that the centralligand-binding cavity of ABCB1 is spatially linked to otherbinding regions located closer to or within the intracellulardomains and/or NBDs [30]. This arrangement might pro-vide access to more hydrophilic ligands from the cytoplasm(Figure 1a), or could provide a functional link betweenligand binding and ATP binding/hydrolysis.

Based on the information inferred from the crystalstructures and biochemical data, the transport mechanismcan be broken down into at least four steps, which together

Drug PDB code Resolution (A) Ref.

– 2HYD 3.00 [47]

– 2ONJ 3.40 [48]

– 3B5W 5.30 [45]

– 3B5X 5.50 [45]

– 3B5Y 4.50 [45]

– 3B5Z 4.20 [45]

– 3B60 3.70 [45]

– 3G5U 3.80 [44]

QZ59-RRR 3G60 4.40 [44]

2 x QZ59-SSS 3G61 4.35 [44]

by the MDs.

sphate; ADP-OV, adenosine diphosphate-orthovanadate; QZ59-RRR, cyclic-tris-(R)-

Figure 2. ABCB1 can use different combinations of flexible side chains in its

ligand-binding cavity to create binding sites for different ligands. The structure

shown is ABCB1 complexed with one molecule of QZ59-RRR (red) or two

molecules of QZ59-SSS (black). Superimposition of the ligand-binding sites:

residues interacting with QZ59-RRR are rendered in red and residues interacting

with QZ59-SSS are rendered in black. The magnified stereo view (inset) reveals

alternative side chain rotamers in the two different drug-bound crystal structures.

Up to 60% of the ABCB1–CPPI interactions in the binding cavity is based on

interactions with Phe and Tyr side chains. The use of different sets of Phe and Tyr

residues in binding of QZ-RRR and QZ59-SSS, and the flexibility of these aromatic

side chains contribute to the specificity of the ligand-binding cavity for different

CPPIs. The N-terminal half of ABCB1 is rendered in green and the C-terminal half is

rendered in blue. This figure was generated in PyMol.

Review Trends in Biochemical Sciences Vol.35 No.1

describe alternating access of the ligand-binding cavity toeither side of themembrane. (i) A ligand enters the bindingcavity from the inner membrane leaflet through the lateralgaps between the two MDs or directly from the cytoplasmand binds the high-affinity ligand-binding site. (ii) TheMDs undergo a conformational ‘switch’ upon bindingand/or hydrolysis of ATP [13,54,55], closing the bindingpocket to the inner leaflet of the membrane and opening itto the outer leaflet. The binding site is thought to reduce itsaffinity for the ligand by decreasing favorable intermole-cular contacts, thereby increasing the off-rate. (iii) Theligand is released at the lateral gap into the outer leafletof the membrane and/or external aqueous environment.(iv) After dissociation of the ligand, the MDs reset to theinward-facing high-affinity state described in (i). It isimportant to note that although the alternating accessmodel for ABCB1 often emphasizes a two-step oscillationbetween an inward-facing conformation and an outward-facing conformation (Figure 1), the conformational changepathways leading from inward-facing to outward-facingand from outward-facing back to inward-facing (collec-tively referred to as ‘transport cycle’) might be non-iden-tical and are most likely to involve multiple intermediatestates. A conformation with disengaged NBDs (and hence,low affinity for nucleotides), as obtained in the ABCB1structure, could represent a compulsory step in this trans-port cycle.

Polyspecificity of the drug-binding cavityThe two reported structures of ABCB1 in complex with oneQZ59-RRRmolecule or two QZ59-SSSmolecules (Figure 2)are consistent with previous observations that ABCB1 andLmrA can bind more than one ligand simultaneously[23,34,56]. The cavity formed by ABCB1 encloses a volumeof approximately 6000 A3 [44] thus providing ample spacefor two drug molecules. The ABCB1–CPPI interactions aredominated by the hydrophobic side chains of non-polar Pheand polar Tyr residues that reside in the apex of the ligand-binding cavity in TMH 1, 6, 7, and 12 (Table 2). These dataagree with previous suggestions regarding the importanceof aromatic residues in ABCB1–ligand binding [57,58]. Pheand Tyr residues account for � 40% and � 60%, respect-ively, of the ABCB1–CPPI interaction as measured byburied surface area in the case of ABCB1 bound to a singleCPPI and two CPPI molecules. The binding of the struc-turally different CPPIs is achieved by the utilization ofdifferent combinations of Phe and Tyr side chains and bythe flexibility of these side chains, allowing them to formrotamers with different orientations in the two non-iden-

Table 2. Summary of interactions between ABCB1 and ligandsa

Transmembrane helix # ABCB1 (QZ5Sav1866, MsbA ABCB1

1 1b –

5 5b Y303

6 6b F332, L335, I

10 7c Q722, F724,

50 11c Y949

60 12c F974, S975, VaResidues within 4 A of the CPPI-type ligands QZ59-RRR and QZ59-SSS are listed. ThebTMH situated in the N-terminal half of ABCB1.cTMH situated in the C-terminal half of ABCB1.

tical drug-bound states (Figure 2). In addition to aromaticside chains, aliphatic and less abundant polar hydrogen-bond donor side chains (Ser, Thr, Gln, Asn) [59] are found,the latter mostly in the walls of the cavity leading to theapex. Hence, structurally diverse ligands can be accom-modated in the binding cavity by stacking and cation–p

interactions and by hydrophobic and hydrogen bondinginteractions, offering a structural explanation for the poly-specificity of this transporter. The chemical nature of the

9-RRR complex) ABCB1 (2 x QZ59-SSS complex)

F71

Y303

336,F339 F332, F339

S725, F728 F724, F728

–

978, A981 L971, F974, I977, L978, G985, S989

background colors are indicative of cartoon rendering in Figures 2 and 4.

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Review Trends in Biochemical Sciences Vol.35 No.1

ABCB1–CPPI interactions is comparable to that observedin structures of other protein–hydrophobic ligand com-plexes. A well known example is provided by soluble multi-drug binding transcriptional regulators, which havemultidrug specificity similar to that of the multidrugtransporters they regulate. In the transcriptional regula-tor QacR from S. aureus, the plasticity of the ligand-bind-ing site is affected by the flexibility and redundancy ofligand-binding residues (including Tyr and Phe sidechains) [60,61] similar to that observed in ABCB1. Inaddition, at least two compounds (the bivalent aromaticdiamidines DB75 and DB359) can bind QacR in more thanone orientation [62].

The ligand-binding residues in ABCB1 that lie inproximity to the CPPI ligands overlap extensively withthose identified in biochemical studies on ABCB1 [63,64],LmrA [42] and MsbA, [28] suggesting that these bacterialtransporters also bind ligands in the apex of the bindingcavity in the inward-facing conformation. However, adirect comparison of the apex regions in the crystal struc-tures of mouse ABCB1 and MsbA, and homology models ofhuman ABCB1, the human hepatic ABCB1 homologABCB4 [65], Sav1866 and LmrA reveals that many hydro-phobic residues are large (Tyr, Phe and Trp) in ABCB1(mouse and human) and ABCB4, but smaller (Gly, Ala,Leu, Ile, Val and Met) in MsbA, Sav1866 and LmrA(Figure 3). Hence, the overlapping ligand specificities ofthese multidrug ABC transporters [26,31,65,66] are notbased on sequence conservations but, instead, rely onfunctional conservation of residues. The size change innon-polar residues in the bacterial transporters and lossof the aromaticity of these residues create steric changesthat alter the affinity for cationic aromatic ligands. ABCB4interacts with the zwitterionic phosphatidylcholine [65],MsbA interacts with the net anionic lipid A anchor oflipopolysaccharides [67] and, more recently, dimeric LmrAwas found to specifically bind anionic cardiolipin [25].These steric changes in the binding cavity might therefore

Figure 3. Stereo views of inward-facing ligand-binding pockets. Views are shown for (

Sav1866 (S. aureus) and (f) LmrA (L. lactis). Aromatic residues (Tyr, Phe and Trp) are co

and all other residues are white. The bacterial models (Sav1866 and LmrA) were based

were based on the crystal structure of ABCB1a. The models were created using Swiss

40

relate to differences in lipid specificity of these ABC multi-drug transporters.

Does helix rotation drive affinity change?Having explored how ligands can bind in the ABCB1ligand-binding cavity, we investigate how the ligand-bind-ing cavity can switch from high affinity in the inward-facing state to low affinity in the outward-facing state,allowing dissociation of the ligand. Fortunately, theinward-facing structures of ABCB1 are complemented bythe outward-facing structures of the homologous bacterialABC transporters MsbA [45] and Sav1866 [47,48]. Bothwere crystallized in nucleotide-bound form, with the drug-binding cavity exposed to the extracellular side. In ouroutward-facing ABCB1 models that were based on thesestructures, the TMHs that carry the majority of residueslining the ligand-binding cavity in the inward-facingABCB1 structure have rotated along the length of theiraxis by up to 908. The CPPI binding residues situated alongTMHs 1, 6, 7 and 12 are forced to follow this rotation(Figure 4). As a result, the amino acid side chains thatpreviously reached into the ligand-binding cavity are nowpartially or completely buried in the MDs, or are evenfacing the lipid bilayer. Thus, when the transporterassumes its outward-facing state in an efflux reactioncoupled to ATP binding and hydrolysis, the ligand-bindingsurface is deprived of the residues that support thestrongest interactions with the ligand. As a result, theligand dissociates and diffuses away. A similar conclusionon helix rotation was reached in a study in which publishedamino acid substitutions in ABCB1 that are associatedwith a change in specificity phenotype [68] were super-imposed on the outward-facing structure of MsbA from S.typhimurium [28]. As observed for Ser289 and Ser290,these residues are partially occluded or face away fromthe drug-binding surface in this outward-facing confor-mation [28]. Biochemical evidence for helix rotationwas obtained by Loo and Clarke [69], who observed that

a) ABCB1a (mouse), (b) ABCB1 (human), (c) ABCB4 (human) (d) MsbA (E. coli), (e)

loured red, hydrophobic residues (Gly, Ala, Leu, Ile, Val and Met) are colored blue

on a full model of MsbA (E. coli) and the mammalian models (ABCB1 and ABCB4)

Model.

Figure 4. The conformational switch in ABCB1 from the inward to the outward-

facing state is accompanied by rotation of the transmembrane helices. Helix

rotations are evident from a comparison of positions of atoms that comprise

helices and bulky amino acid side chains. During the rotation, side chains

important for ligand binding to the inward-facing conformation (i) move away

from the binding cavity. As favorable protein–ligand interactions are disrupted, the

reduced binding affinity in the outward-facing conformation (ii) allows dissociation

of the ligand from the binding cavity. The inward-facing and outward-facing

conformations of the ABCB1 MDs are seen from the outside of the cell. Loops and

NBDs are omitted for clarity. Residues involved in ligand binding are shown as

sticks. Residues residing on TMHs 1, 6, 7 and 12 are shown in red. Residues

contributed from other helices are rendered in green. Arrows refer to helix rotation

from the inward to the outward state. To generate the outward-facing view, the

residues from ABCB1 were superimposed onto the outward-facing structure of S.

typhimurium MsbA using Modeller. This figure was generated in PyMol.

Review Trends in Biochemical Sciences Vol.35 No.1

alteration of thiol cross-linking between TMHs 6 and 12 inABCB1 by ATP hydrolysis required rotation of one helix orboth. In considering the structural similarity betweenABCB1, LmrA, Sav1866 and MsbA, it is possible thatthe mechanism involving helix rotations is conservedamong multidrug ABC transporters.

Concluding remarksThis review summarizes how the insights gained by thefirst structures of ABCB1 complement the establishedbiochemical understanding of this transporter and itsbacterial homologs. Yet these structures also allow usto ask more questions. The CPPI ligands QZ59-RRRand QZ59-SSS clearly inhibit ABCB1-ATPase activityin the detergent-solubilized form as well as ABCB1-mediated transport and drug resistance in intact cells,suggesting that these inhibitors are not transported orthat they are transported at a very low rate [44]. However,the CPPI inhibitors are bound to a ligand-binding cavityin ABCB1 that can also bind ligands that are rapidlytransported. What distinguishes the inhibitor from thetransported ligand? Do inhibitors and ligands bind differ-ent sets of residues? If the inward-facing V-shapedABCB1 conformation is part of the transport cycle, couldthe binding of one QZ59-RRR molecule or two QZ59-SSSmolecules prevent the tight dimerization of half-trans-porters necessary for propagation of this cycle? If thistight dimerization would usually lead to the formation ofan asymmetric closed conformation of the two half-trans-porters, would two transported ligands be translocatedsimultaneously or in a sequential or alternating fashion[23] that is dictated by the mechanism of catalysis at theNBDs [13,70]? Could these inhibitors sterically hinder thehelix rotation(s) required for their release at the oppositeside of the membrane? The answers to these questionsmight hold important clues to our understanding of the

mechanisms of multidrug ABC exporters and the futuredevelopment of modulators of these transport systems,which would have an enormous impact on the efficacy ofchemotherapeutic treatment of infectious diseases andcancer. Progress will require further structural and bio-chemical characterizations of a variety of transport cycleintermediates of mouse and human ABCB1 and theirbacterial homologs that provide useful and accessiblemodels.

Disclosure statementThe authors have declared that no competing interestsexist.

AcknowledgmentsWe apologize to all colleagues whose work was not cited in this briefreview. We thank James Smith and Alan Senior for discussions. Our workwas supported by the Biotechnology and Biological Sciences ResearchCouncil (BBSRC) (to H.W.v.V.), and the Jasper L. and Jack DentonWilson Foundation (to I.U.), and by grants from the US Army (W81XWH-05-1-0316), NIH (GM61905), and the Skaggs Chemical BiologyFoundation (to G.C.).

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