Structures of ABCB10, a human ATP-binding cassettetransporter in apo- and nucleotide-bound statesChitra A. Shintrea,1, Ashley C. W. Pikea,1, Qiuhong Lia,2, Jung-In Kima,3, Alastair J. Barra,4, Solenne Goubina,Leela Shresthaa, Jing Yanga, Georgina Berridgea, Jonathan Rossa, Phillip J. Stansfeldb, Mark S. P. Sansomb,Aled M. Edwardsc, Chas Bountraa, Brian D. Marsdena, Frank von Delfta, Alex N. Bullocka, Opher Gileadia,Nicola A. Burgess-Browna, and Elisabeth P. Carpentera,5

aStructural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, OX3 7DQ, United Kingdom; bStructural andComputational Bioinformatics Unit, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, United Kingdom; and cStructural GenomicsConsortium, University of Toronto, Toronto, ON, Canada M5G 1L7

Edited* by Wayne A Hendrickson, Columbia University, New York, NY, and approved April 24, 2013 (received for review October 2, 2012)

ABCB10 is one of the three ATP-binding cassette (ABC) transportersfound in the innermembraneofmitochondria. InmammalsABCB10 isessential for erythropoiesis, and for protection of mitochondriaagainst oxidative stress. ABCB10 is therefore a potential therapeutictarget for diseases in which increased mitochondrial reactive oxygenspecies production and oxidative stress play a major role. The crystalstructure of apo-ABCB10 shows a classic exporter fold ABC trans-porter structure, in an open-inwards conformation, ready to bindthe substrate or nucleotide from the inner mitochondrial matrixor membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATPanalogs, in contrast to other transporter structures which adopt anopen-outwards conformation in complex with ATP. The three com-plexes of ABCB10/ATP analogs reported here showed varying de-grees of opening of the transport substrate binding site, indicatingthat in this conformation there is some flexibility between the twohalves of the protein. These structures suggest that the observedplasticity, together with a portal between two helices in the trans-membrane regionofABCB10, assist transport substrate entry into thesubstrate binding cavity. These structures indicate that ABC trans-porters may exist in an open-inwards conformationwhen nucleotideis bound. We discuss ways in which this observation can be alignedwith the current views on mechanisms of ABC transporters.

ABC mitochondrial erythroid | X-ray crystallography |human membrane protein structure | nucleotide complex | cardiolipin

ATP-binding cassette (ABC) transporters move small mole-cules, ions, hormones, lipids, and drugs across cell membranes,

and have diversified to act as ion channels and components ofmultiprotein complexes (1, 2). ABC transporters have critical rolesin many diseases, including juvenile diabetes (3), cystic fibrosis (4),and drug resistance in cancer (5). ABC transporters are ubiquitousproteins, bacteria having several hundred examples and humanshaving 48 homologs (6). These diverse proteins share a commonarchitecture with two nucleotide binding domains (NBDs) and twotransmembrane domains (TMDs). The NBDs bind and hydrolyzeATP, providing the energy to move substrates across membranesagainst a concentration gradient. The TMDs are more diverse, withseveral possible folds in bacteria, which provide binding sites fora broad range of substrates (reviewed in refs. 1 and 2). ABC trans-porters function by an alternating access mechanism, where theTMD substrate binding sites alternate between outward- and in-ward-facing conformations (7). Structures ofABC transporterswiththe exporter fold have been obtained without bound nucleotide inthe open-inwards conformation (8–10) and with nucleotides boundin the open-outwards conformation (9, 11). However, the role ofconformational changes, the mechanism by which ATP hydrolysisdrives transport and the sequence of substrate and nucleotidebinding remain controversial (2).ABCB10 (also known as ABC mitochondrial erythroid, ABC-

me, mABC2, or ABCBA) is one of the three ABC transporters

found in the inner membrane of human mitochondria, with theNBDs inside the mitochondrial matrix (12, 13). Mitochondriasynthesize ATP, a process that produces toxic reactive oxygenspecies, which damage mitochondrial DNA; they are also the siteof synthesis of metabolites, such as heme and lipids. ABCB10expression is induced during erythroid differentiation and over-expression increases hemoglobin synthesis (12). However, ABCB10is also expressed in many nonerythroid tissues, suggesting addi-tional roles not related to hemoglobin synthesis (13, 14). Inter-estingly, recent reports identified ABCB10 as a key player inprotection against oxidative stress and processes intimately re-lated to mitochondrial reactive oxygen species generation, suchas cardiac recovery after ischemia and reperfusion (15, 16).ABCB10 knockout mice die at 12.5-d gestation, and were anemicat day 10.5, during a period where primitive erythropoiesis wouldnormally occur (14). A potential role for ABCB10 would be ex-port of a heme biosynthesis intermediate, in which case ABCB10−/−

mice would not be able to synthesize hemoglobin. However,ABCB10−/− mouse embryos do still produce a minor populationof hemoglobinized erythroid precursors, so a low level of hemo-globin synthesis still occurs in the absence of ABCB10. Anotherrole proposed for ABCB10 or potential homologs is stabilizationof the iron transporter mitoferrin-1 (SLC25A37) (17, 18). A studyof the yeast homolog of ABCB10 multidrug resistance-like 1(41% identity) (19) suggested that the substrates of ABCB10 maybe peptides of 6–20 amino acids that result from digestion ofproteins by the m-AAA protease in the mitochondrial matrix(20). To improve our understanding of ABCB10 function andto facilitate the identification of substrates, we have solved thecrystal structure of human ABCB10 in complex with nucleotideanalogs and in a ligand-free, apo form. These structures have given

Author contributions: C.A.S., A.C.W.P., A.J.B.,M.S.P.S., A.M.E., C.B., B.D.M., A.N.B., O.G., N.A.B.-B.,and E.P.C. designed research; C.A.S., A.C.W.P., Q.L., J.-I.K., A.J.B., S.G., L.S., J.Y., G.B., J.R., P.J.S.,F.v.D., O.G., N.A.B.-B., and E.P.C. performed research; C.A.S., A.C.W.P., P.J.S.,M.S.P.S., A.M.E., C.B.,and E.P.C. analyzed data; and C.A.S., A.C.W.P., and E.P.C. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Freely available online through the PNAS open access option.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org [PDB ID codes 4AYT (rod form A), 4AYX (rod form B),4AYW (plate form), and 3ZDQ (nucleotide-free rod form)].1C.A.S. and A.C.W.P. contributed equally to this work.2Present address: Section of Structural Biology, Institute of Cancer Research, ChesterBeatty Laboratories, London SW3 6JB, United Kingdom.

3Present address: Institute of Molecular Biology and Biophysics, Eidgenössiche TechnischeHochschule Zürich, 8093 Zürich, Switzerland.

4Present address: Department of Human and Health Sciences, School of Life Sciences,University of Westminster, London W1W 6UW, United Kingdom.

5To whom correspondence should be addressed. E-mail: liz.carpenter@sgc.ox.ac.uk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1217042110/-/DCSupplemental.

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us an unexpected conformation for nucleotide analog complexesand new insights into the transport cycle for ABC transporters ofthe exporter fold.

ResultsStructure ABCB10 in Complex With Nucleotide Analogs and WithoutBound Substrate. To solve the structure of ABCB10, we expressedthe protein in insect cells without the N-terminal mitochondrialtargeting sequence as this improved protein yields. Two constructsof ABCB10 were purified in the detergent dodecyl maltoside

(DDM)with the addition of either cholesteryl-hemisuccinate (CHS)or the mitochondrial lipid cardiolipin (CDL). We obtained crystalsof ABCB10 both without nucleotide analogs and in the presence ofthe ATP analogs adenosine-5′(βγ-imido)triphosphate (AMPPNP)or β-γ-methyleneadenoside 5′-triphosphate (AMPPCP) (Table S1).Initially we solved the structure using plate-form crystals, whichwere phased by isomorphous replacement using a single mercuryderivative. We subsequently obtained rod-form crystals that weresolved by molecular replacement. The structure solution methodsare summarized in Materials and Methods and are described indetail in SI Materials and Methods. Crystallographic statistics areavailable in Table S1 and the quality of maps is shown in Fig. S1.The overall fold of ABCB10 (Fig. 1A) is common to most eu-

karyotic ABC transporters and some bacterial exporters, and waspreviously observed for the bacterial multidrug transporter Sav1866(11), the lipid flippase MsbA (9), and the mouse multidrug effluxprotein P-glycoprotein (mP-gp) (8) and its Caenorhabditis eleganshomolog (ceP-gp) (10). The ABCB10 fold consists of a shortN-terminal α-helix, lying parallel to the plane of the membrane,followed by six long transmembrane α-helices (TMH) that tra-verse the lipid bilayer and project a further 30 Å into the mito-chondrial matrix. The two monomers are interconnected by adomain swap where TMH4 andTMH5 interact with TMH1–3 andTMH6 from the other half of the dimer (Fig. 1B). The TMD isconnected to the C-terminal NBD by an extended linker. The C-terminal domain adopts a classic NBD fold for anABC transporter,with a RecA-like core subdomain, an α-helical subdomain, anda disordered C terminus. ABCB10 is a homodimeric half trans-porter and, in both the crystal forms, there is one monomer in theasymmetric unit. The dimer is generated by a crystallographictwofold axis, so the two halves of the dimer form a symmetricalstructure (Fig. 1B).

Fig. 1. Structure of ABCB10 in complex with the nonhydrolysable nucleo-tide analog AMPPCP, showing that ABCB10 is in an open conformation, evenwhen it is bound to nucleotide analogs. Cartoon representations of theABCB10/AMPPCP complex monomer (A) and homodimer (B) as seen in the rod-form B crystal structure. The structures have a single monomer in the asym-metric unit, the dimer is generated by a crystallographic twofold.

Fig. 2. Comparison of the structures of the ABCB10 homodimer in the absence (apo) and presence of bound nucleotide analogs, with the structures of ceP-gp inthe open-inwards conformation [PDB ID code: 4F4C (10)] and Sav1866 [PDB ID code 2HYD (11)] in the open-outwards conformation. Transporters are viewedperpendicular to their (pseudo) twofold symmetry axes. (B) Alignment of NBDs of the structures shown in A. viewed looking toward the membrane. The ABCB10monomers (blue/purple and orange/red respectively), nucleotides (green), and the NBD’s C-loop (cyan) are highlighted. The black lines/circles below each NBDpair indicate the translation required to bring the NBDs in the closed conformation for catalysis [the distance is the separation between the nucleotideγ-phosphate (green) and the C-α of the first glycine in the catalytic C loop of the adjacent NBD (cyan)]. An additional rotational component is also required forproper alignment of the NBDs in the closed state. Where trinucleotide is not present in the structure, the position of the γ-phosphate has been inferred bysuperposition of the AMPPCP complex. The C-terminal extension in the Sav1866 structure has been omitted for clarity.

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Although ABC transporters of the exporter family have thesame overall fold, they adopt radically different conformationsduring the transport cycle as they move from open-inwards to anopen-outwards state, allowing the transporters to bind substrateon one side of the membrane and release the substrate on theother side. In the absence of nucleotide analogs, an open-inwardsconformation has been observed where the NBDs are not incontact and the molecule forms an extensive substrate bindingcavity facing the NBDs. This is the conformation we observe forour nucleotide-free ABCB10 structure (Fig. 2A) and was pre-viously observed for MsbA (9), mP-gp (8), and ceP-gp (10) (Fig.2A). The alternate open-outward conformation was found forcomplexes of ADP, ADP/vanadate, and AMPPNP with Sav1866(11) (Fig. 2A) and MsbA (9). In the latter conformation, theNBDs are closely packed with two nucleotides sandwiched be-tween the NBDs at the interface between the RecA-like coresubdomain of one NBD and the α-helical subdomain of thesecond (Fig. 2B). In addition, a heterodimeric ABC transporter,TM287/288, from the thermophilic bacterium Thermotoga maritimahas been solved in an intermediate open-inwards conformationwith AMPPNP bound only to the noncanonical, high ATP af-finity site, not to the catalytic site. In this case the NBDs are incontact at the high-affinity ATP binding site, but not at thecatalytic site (21).Unexpectedly, ABCB10 in complex with nucleotide analogs

is in an inward-facing conformation with the NBDs separated,similar to the conformation without bound nucleotide (Fig. 2A).We observe clear density for both nucleotide and the magnesiumion in the higher-resolution rod-form crystals with bound AMPPCP.In the lower resolution plate-form crystals with AMPPNP, theNBDs are more disordered and have higher B factors, and herethe density is only clear for the base, ribose ring and two phos-phates (Fig. S1D–F). The open-inwards conformation is observedin two crystal forms with unrelated packing (Fig. S2) and in sev-eral crystals for each crystal form. There is clearly considerableflexibility in the structure, different crystals giving structures withvariation in the extent of opening or closing of the open con-formation (Fig. 2), (Fig. S3 and Note 1 in the SI Materials andMethods provide further information on the differences betweenopen conformation ABCB10 structures). However, in no case didwe observe a change to the open-outwards conformation seen inMsbA and Sav1866 or the half-closed NBDs seen the TM287/288structure (Fig. S4). The flexibility we observe for the ABCB10structures is in line with that observed for other ABC transport-ers in the open conformation, such as mP-gp (8) and MsbA (9).Molecular dynamics simulations on the two high-resolution nu-cleotide complex structures of ABCB10, embedded in a phospho-lipid bilayer, indicated that overall the structure is stable during a100-ns simulation (Fig. S5 A–C), the only change in the structureoccurring around TMH6, which has a tendency to unwind betweenGly446 and Gly447 at the glycine-rich sequence G458LGAGG inall four simulations (Fig. S5D).

ABCB10 Nucleotide Binding Site. The nucleotide binding site inABCB10 is only partially formed. In earlier ABC transporter nu-cleotide complexes in the open-outwards conformation, the NBDsare tightly packed together, with two nucleotides sandwiched at theinterface, each interacting with both NBDs. However, in theABCB10/AMPPCP structure the NBDs are separated to varyingdegrees (Fig. 2B) and each NBD interacts with a single nucleotide(Fig. 3A). ABC transporters have highly conserved nucleotidebinding sites with a series of conserved motifs that interact with thenucleotide. The interactions between the nucleotide and theWalker A motif, A-loop tyrosine (Tyr501), and the Q-loop gluta-mine (Gln575) from one NBD are very similar to those in otherABC transporter structures (Fig. 3C), but the interaction with theABC transporter consensus LSGGQ sequence (C-loop) fromthe other NBD is absent (Fig. 3A). This finding is reminiscent of

the interactions observed for isolated monomeric NBDs with ADP(22), bacterial maltose importer with AMPPNP, and maltose (23)and one site of TM287/288 (21). In both nucleotide-bound andnucleotide-free forms of ABCB10 the Walker B motif glutamate(Glu659) adopts an unusual position, rotated 180° away from itsexpected orientation adjacent to the γ-phosphate. This conformationhas been observed in isolated monomeric NBDs for ABCB6 (22).The His-loop histidine (His690) side-chain is partially disordered(not modeled in the rod-form A structure) and is further awayfrom the γ-phosphate site than is usually observed in ABCtransporters.We would expect these residues to move to the morecommonly observed positions once the NBDs come together.In the absence of nucleotide the ABCB10 NBDs have a similar

conformation, except in the region of the Walker A motif, resi-dues 530–534 (Fig. 3D), which form an additional turn of theWalker A helix when there is no nucleotide bound. In the nu-cleotide complexes these residues form an extended loop struc-ture that interacts with the β/γ-phosphates of bound nucleotide(Fig. 3C). This change in the local conformation of the nucleo-tide binding site is observed in some (22) but not all (8) nucle-otide-free ABC transporter NBDs.The classic ATP switch mechanism suggests that when two

nucleotides bind to an ABC transporter dimer, it should convert tothe open-outwards conformation (24), but if only one of the two

Fig. 3. Interactions of nucleotides with the ABCB10 inward-facing confor-mation. (A) Schematic representation of NBDs of ABCB10 homodimer in thehighest-resolution structure (rod form A) viewed looking from the TMD.AMPPCP (green/orange) are bound to each NBD but do not make inter-NBDinteraction with the ABC transporter consensus sequence LSGGQ C-loopmotif(cyan). (B) Individual NBD viewed looking from adjacent NBD. NBD (pink) withABC Transporter nucleotide binding signature motifs colored as in C. TMD(gray) and coupling helices (CH1, orange; CH2#, purple) are highlighted. OmitFo − Fc density for AMPPCP/Mg2+ (blue mesh) is shown contoured at 3 σ.Dotted box indicates zoomed region in C and D. Detailed view of nucleotidebinding site in rod form A/AMPPCP complex (C) and nucleotide-free form (D).Oxygen atoms are colored red, nitrogen atoms blue, phosphate atoms orangeand carbon atoms are colored according to the location of the atom: TheAMPPCP carbon atoms are shown in green. The conserved NBD sequencemotifs are coloredWalker A (yellow), Walker B (light blue), A-loop (pale cyan),Gln-loop (red), His-loop (light green), coupling helix 1 (CH1, orange), andcoupling helix 2 (CH2, magenta). The C-loop (cyan) is not visible as it is morethan 16 Å away from the nucleotide binding site in this conformation. Resi-dues/secondary structure elements marked with a pound symbol (#) denoteregions contributed by homodimer partner. The side-chain of His690 is disor-dered and has not been modeled in rod form A.

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nucleotide binding sites has nucleotide bound, then the structurewould remain in the open-inwards conformation. If this hypothesiswere true, then for our homodimeric structures with a singlemonomer in the asymmetric unit, we would expect to see an oc-cupancy for the nucleotide of 50% or less. However, this result isnot what we observe; refinement of the highest-resolutionABCB10 structure with fixed occupancies for the nucleotide andthe magnesium ion of either 50%, 80%, or 100%, gave B factorsfor the nucleotide that were 30 Å2 below, similar to, or 30 Å2

above the average B factor for residues adjacent to the boundnucleotide. The occupancies of the nucleotide and Mg2+ aretherefore in the 80–100% range, confirming that the majority ofdimers would have two nucleotides bound. This structure, there-fore, represents an open-inwards conformation with nucleotidebound to both nucleotide binding sites in the majority of molecules.

ABCB10 ATPase Activity and Inhibition by Nucleotide Analogs. Be-cause the open-inwards, nucleotide-bound conformations are un-expected, we investigated whether the ABCB10 ATPase activityand inhibition of this activity by nucleotide analogs differed fromthose of other ABC transporters. ABC transporters use the bindingand hydrolysis of ATP to power the movement of substrates. Sur-prisingly, many ABC transporters exhibit a basal ATPase activity invitro in the absence of transport substrate, which is stimulated be-tween 2- and 10-fold when a transport substrate is added (25–27).ABCB10 showed basal ATPase activity, with apparent kineticparameters similar to those of other ABC transporters (28, 29),when it was purified in DDM and either CHS or CDL, with orwithout reconstitution into liposomes (Table 1, Fig. S6A andB, andSI Materials and Methods). It is clear that ABCB10 is active both inmicelles and in proteoliposomes (Fig. S6 A and B). Small increasesinKcat/Km, of the order of fourfold on reconstitution are a commonfeature of many ABC transporters (29–31). The basal ATPase ac-tivity is inhibited by vanadate (Fig. S6 A and B). CDL does notstimulate the ATPase activity when added during reconstitution(Fig. S6C), suggesting that it is unlikely that CDL is transported byABCB10. Mutation of the conserved putative catalytic Glu659 toglutamine in the nucleotide binding site of ABCB10 gave proteinwith no detectable activity (Fig. S6B andE), as is observed for otherABC transporters (32, 33). The first construct used for crystalliza-tion had a PCR-derived mutation, which converted Arg691 toa histidine. The R691Hmutation led to a substantial loss of activityand ATPase activity was restored when the Arg691 was reintro-duced (Fig. S6D). The significance of this observation is discussed inNote 3 of SI Materials and Methods.The nucleotide analog complex structures presented here have

AMPPNP or AMPPCP bound to the NBDs. We investigated theinhibition of the ABCB10 ATPase activity by these nucleotideanalogs (Fig. S6F). AMPPCP and AMPPNP have IC50s of 2.3 ±0.9 mM and 1.1 ± 1.1 mM, respectively, measured with 2 mMATP. Using the Km,app of 0.2 mM measured for this proteinsample, the apparent Ki for AMPPCP is of the order of 0.2 mMand for AMPPNP is 0.1 mM. The affinity of ABCB10 for these

nucleotide analogs is therefore of the same order-of-magnitudeas its affinity for ATP, suggesting that the interactions observedfor the analogs would be similar to those for ATP.

Portal Between Two Transmembrane Helices Could Be a Route forSubstrate Entry. Lipid and detergent molecules are clearly visibleinteracting with the outer surface of the membrane-spanning re-gion in all four ABCB10 structures (Fig. S7). Intriguingly in therod-form crystals, TMH1 and TMH2 are separated, with elon-gated electron density between these helices, which we have at-tributed to an alkyl chain of a CDL molecule (Fig. 4 A and B). Thislipid chain contacts both the internal cavity and the outer surfaceof the protein. The portal through which this CDL passes links theinternal TMD cavity and the center of the lipid bilayer. In contrast,in the plate-form crystals these helices are packed tightly together,with no opening between them (Fig. 4C). In the rod-form crystalsthere are three residues which form crystal contacts to the loopjoining TMH1 and TMH2, but there are no crystal contacts in thetransmembrane region involving these helices, so it is unlikely thatthis change in conformation is induced by the crystal contactsobserved in the rod-form crystals. This portal could providea route of entry for a hydrophobic or amphipathic substrate fromthe membrane into the binding cavity.

Conserved Residues in the TMD Suggest a Substrate Binding Site forABCB10. Alignment of the protein sequences of ABCB10 paralo-gues in 80 organisms highlights conserved patches that are ex-posed on the inner surface of the transporter (Fig. 5 and Fig. S8).The external surface of ABCB10 is not conserved, suggestingthat ABCB10 has a role in substrate transport rather than forminga complex with another protein, which would require a conservedexternal surface. The NBD signature motifs are highly conserved,together with a patch formed by residues at the N-terminal endof TMH3 and the C-terminal end of TMH6. This latter clusterinteracts closely with conserved residues in the domain-swappedTMH4 (Q344/A348) in the open-out conformation. A third clusteris located on the concave surface formed by the TMD. There aretwo conserved motifs, (N/I)xxR located in the center of TMH2 andNxxDGxR at theN terminus of TMH3B, which form a patch on theinner surface of the TMD cavity (Fig. 5 B–D). These residues forman ABCB10 signature sequence and, based on their location, wesuggest that they could form part of the substrate binding site foran amphipathic substrate. If the outward-facing conformation ofABCB10 resembles that of Sav1866 (11), then the two helices

Table 1. Kinetic parameters for the basal ATPase activityof ABCB10

Lipid* Recon† n‡

Km, app

(ATP) mMVmax, app

(nmol Pi min−1·mg−1) Kcat/KmM-1·s

-1

CHS − 2 0.18 ± 0.02 51 ± 1.1 2.2CHS + 10 0.3 ± 0.08 319 ± 27 8.2CDL − 2 0.5 ± 0.2 106 ± 18 1.6CDL + 3 0.19 ± 0.03 181 ± 9.8 6.7

*Protein purified in DDM, with the addition of either CHS or CDL.†Protein in detergent (−) or reconstituted into proteoliposomes (+).‡n = number of independent purifications.

Fig. 4. ABCB10 has cardiolipin and detergent bound to the transmembranehelices and a portal between helices TMH1 and TMH2, which is open in the rodcrystal form and closed in the plate-form crystals. (A) Overview of the ABCB10structure showing the location of lipid (magenta) and detergent (green)binding sites. (B) Molecular surface representation of the TMD in rod form Acrystals, with lipid and detergent molecules shown in magenta and green andthe portal between TMH1 and TMH2 indicated with a dotted line. In the rod-form structures TMH1 and TMH2 are loosely packed revealing a 7 Åwide× 30Ålong portal connecting the central cavity of the TMDs with the membraneenvironment. The portal is occupied by a CDL alkyl chain (magenta). (C) In theplate form crystals TMH1 and TMH2 are packed closer together, with the portalclosed. Structures are viewed in the same orientation as in A.

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TMH2 and TMH3 would be substantially further apart in theopen-outward structure, changing the substrate binding site andtherefore releasing the substrate into the intermembrane space.

DiscussionABCB10 Structure and ABC Transporter Mechanism. Our four struc-tures of ABCB10 in complex with nucleotide analogs and withoutbound nucleotide give snapshots of ABCB10 before substratebinding and in a conformation where ATP has bound, and theprotein is awaiting the transport substrate. ABCB10 remains anorphan transporter, one for which we do not yet know the transportsubstrates. Our structures do, however, suggest a potential entryroute for an amphipathic substrate and a potential binding site fortransport substrates.The generally accepted ATP switch model for ABC transporter

function (24) proposes that binding of nucleotide to the protein/substrate complex is the trigger for conversion from the open-in-wards to the open-outward conformation, thus driving transport ofthe substrate across the membrane. A similar mechanism, but with

the nucleotide binding before the substrate, was suggested for thetransporter associated with antigen processing (34) and alternativebinding sequences have also been proposed for P-gp (35). Alter-nating catalytic site mechanisms involving asymmetric binding andhydrolysis have also been put forward (reviewed in ref. 36).Our structures suggest an adaptation of the accepted ATP

switch mechanism in which either transport substrate or nucle-otide could bind first (Fig. 6). These ABCB10 nucleotide analogcomplex structures show that nucleotides can bind in the absenceof a transport substrate, an observation that is in line with thefact that many ABC transporters, including ABCB10, have a basalATPase activity, performing the ATPase reaction in the absenceof transport substrate. ABC transporter NBDs must thereforebe capable of binding ATP and coming together in a productivecomplex for ATP hydrolysis, even when there is no transportsubstrate present.Although our structures indicate that ABC transporters can

bind nucleotide in the absence of transport substrate, it has alsobeen shown that many ABC transporters bind transport sub-strate and inhibitors in the absence of nucleotide, as has beenobserved for P-gp (8) and transporter associated with antigenprocessing (37). We therefore propose that ABC transporterscould bind either nucleotides or transport substrate first, eachbinding initially to one face of open-inwards conformation. Oncethe nucleotides or transport substrate binds to one face of thecomplex, the NBDs or TMDs could come together in a productiveorientation, forming complete binding sites with interactions forthe substrate molecules with both halves of the TMDs and forthe ATPs with both NBDs (Fig. 6). Because the rate of ATPturnover is often higher in the presence of the transport substrate,occupation of the transport substrate binding site must promoteformation of the ATP hydrolysis-competent closed NBD con-formation. The NBDs coming together in the presence of boundtransport substrate would then trigger reorganization of theTMDs into the open-outwards conformation, disrupting thetransport substrate binding site and forcing the substrate todissociate on the other side of the membrane. One or both of thenucleotides would then be hydrolyzed and dissociate, allowingthe structure to return to the inward-facing conformation. Al-though our structures are symmetrical, having a monomer in theasymmetric unit, this does not preclude the possibility that cer-tain steps in the mechanism involve asymmetry in the nucleotidebinding site, with hydrolysis occurring in only one site, as hasbeen shown for other ABC transporters (36). These ABCB10structures therefore provide fresh insights into ABC transporterfunction, in particular providing a unique example of exporterfold ABC transporters in complex with nucleotide analogs, in anopen-inwards conformation. The findings suggest that nucleotide

Fig. 5. Sequence conservation of residues inABCB10. Conservation is mapped onto a molecularsurface representation of the concave inner cavity(A) and convex outer surface (B) of ABCB10. Themolecular surface shown is a composite represent-ing one-half “leaflet” of the transporter dimer,comprising residues from TMH1–TMH3 and TMH6-NBD in one monomer and TMH4/5 from the secondmonomer (residues 311–424). Residues are coloredaccording to sequence conservation, invariant (darkblue), highly conserved (slate; 2–4 related aminoacids), and moderately conserved (pale blue; 3–8related amino acids). Sequence conservation wascalculated based on an alignment of eighty ABCB10homologs (human to yeast) using the CONSURFserver (38). The conserved patch in the TMD be-tween TMH2/TMH3B adjacent to the portal region(indicated in red) defines a unique ABCB10 signature sequence. (C and D) Perpendicular views of residues defined by ABCB10 signature sequence (Asn229,Arg232, Asn289, Asp295, and Arg295). All of the side-chains project toward the lumen of the transporter. Side-chains are shown along with a semitransparentmolecular surface. (E) Conserved signature sequence defined by residues located in central portion of TMH2 and at the N-terminal end of TMH3B.

2 ATP

Substrate2 ATP

Substrate 2 ADP

2 Pi

90°

#

View rotated 90°

Fig. 6. Overview of the steps proposed for the transport cycle of ABCB10and other ABC transporters of the exporter family. The TMDs are coloredblue and orange, with their associated NBDs colored purple and red. ATP isshown in green, ADP in gray and the transport substrate in yellow. An as-terisk (*) and pound symbol (#) indicate the conformations observed fornucleotide-bound and nucleotide-free ABCB10, respectively.

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binding can occur before transport substrate binding and that itis therefore likely that the order in which these molecules bindis not fixed. Binding of the transport substrate does, however,promote the formation of the conformation in which the NBDscome together, converting the TMDs to the open-outwards con-formation, thus leading to a stimulation of the ATPase reactionand transport of the substrate.The key role of ABCB10 in erythropoiesis and relief of oxi-

dative stress suggests that this transporter could be an interestingcandidate to explain a frequently observed adverse effect ofdrugs and early clinical inhibitors in red blood cell formation,resulting in anemia or decreased recovery of cardiac functionafter ischemia/reperfusion. The future identification of ABCB10substrates and binding studies of inhibitors and drugs that havebeen associated with these side effects will facilitate identifica-tion of the function of ABCB10. Such studies would also giveinsight into the role of ABCB10 in conditions causing increasedmitochondrial oxidative stress, such as aging, anemia, cardiacischemia/reperfusion, or neurodegenerative diseases and its po-tential as a target for pharmaceutical intervention.

Materials and MethodsThe complete methods are presented in SI Materials and Methods. Forstructure and function studies we expressed ABCB10 in insect cells usingbaculovirus vectors. We expressed ABCB10 with both N- and C-terminal His-tags with the mitochondrial targeting presequence (mTP) removed, eitherby deletion of the N-terminal 151 residues or removal of residues 6–126. Weextracted and purified ABCB10 in the detergent DDM with the addition ofeither CHS or CDL by cobalt affinity and size-exclusion chromatography. For

functional studies we reconstituted ABCB10 into liposomes containing thesynthetic lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine (POPE) without theaddition of CHS or CDL.

We obtained plate-form crystals with both ABCB10 constructs, purified inDDM and CHS, without addition of nucleotide and with the nucleotide analogsAMPPNP or AMPPCP. All nucleotide analog stocks were prepared with equi-molar magnesium chloride. Crystals were grown at 20 °C using the sitting-dropvapor-diffusion method. Diffraction data were collected on beamline I24 atDiamond Light Source. Crystals grown with AMPPNP diffracted anisotropicallybeyond 3.3 Å and were used to solve the structure with phases from a singlemercury derivative (Fig. S1 and Table S1). The chain trace was confirmed withdata from selenomethionine-labeled ABCB10 crystals. Rod-shaped crystalswere obtained without nucleotide and with AMPPCP from protein purified inthe presence of CDL. The rod-form crystals diffracted to at least 2.9 Å andwere phased by molecular replacement using an initial model derived fromthe plate form. ATPase activity assays and molecular dynamics simulationsmethods are described in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Robert Tampé and Peter Henderson forhelpful discussions and Stefan Knapp for critical reading of the manuscript;Richard Callaghan for help with establishing the ATPase assay; Tobias Krojer,Melanie Vollmar, and Joao Muniz for assistance with crystal screening; thestaff at Diamond Light Source and, in particular, the microfocus beamlineI24 for assistance with crystal screening and data collection. The StructuralGenomics Consortium is a registered charity (no. 1097737) that receivesfunds from the Canadian Institutes for Health Research, Genome Canada,GlaxoSmithKline, Lilly Canada, the Novartis Research Foundation, Pfizer,Takeda, AbbVie, the Canada Foundation for Innovation, the Ontario Min-istry of Economic Development and Innovation, and the WellcomeTrust (092809/Z/10/Z).

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