Structures of ABCB10, a Human ATP-Binding Cassette Transporter in Apo- and Nucleotide-Bound States

Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states

Chitra 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 and Computational Bioinformatics Unit, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, United Kingdom; and cStructural Genomics Consortium, 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) transporters found in the inner membrane of human mitochondria, with the found in the inner membrane of mitochondria. In mammals ABCB10 is NBDs inside the mitochondrial matrix (12, 13). Mitochondria essential for erythropoiesis, and for protection of mitochondria synthesize ATP, a process that produces toxic reactive oxygen against oxidative stress. ABCB10 is therefore a potential therapeutic species, which damage mitochondrial DNA; they are also the site target for diseases in which increased mitochondrial reactive oxygen of synthesis of metabolites, such as heme and lipids. ABCB10 species production and oxidative stress play a major role. The crystal expression is induced during erythroid differentiation and over- structure of apo-ABCB10 shows a classic exporter fold ABC trans- expression increases hemoglobin synthesis (12). However, ABCB10 porter structure, in an open-inwards conformation, ready to bind is also expressed in many nonerythroid tissues, suggesting addi- the substrate or nucleotide from the inner mitochondrial matrix tional roles not related to hemoglobin synthesis (13, 14). Inter- or membrane. Unexpectedly, however, ABCB10 adopts an open- estingly, recent reports identified ABCB10 as a key player in inwards conformation when complexed with nonhydrolysable ATP protection against oxidative stress and processes intimately re- analogs, in contrast to other transporter structures which adopt an lated to mitochondrial reactive oxygen species generation, such open-outwards conformation in complex with ATP. The three com- as cardiac recovery after ischemia and reperfusion (15, 16). plexes of ABCB10/ATP analogs reported here showed varying de- ABCB10 knockout mice die at 12.5-d gestation, and were anemic grees of opening of the transport substrate binding site, indicating at day 10.5, during a period where primitive erythropoiesis would that in this conformation there is some flexibility between the two normally occur (14). A potential role for ABCB10 would be ex- − − halves of the protein. These structures suggest that the observed port of a heme biosynthesis intermediate, in which case ABCB10 / plasticity, together with a portal between two helices in the trans- mice would not be able to synthesize hemoglobin. However, − − membrane region of ABCB10, assist transport substrate entry into the ABCB10 / mouse embryos do still produce a minor population substrate binding cavity. These structures indicate that ABC trans- of hemoglobinized erythroid precursors, so a low level of hemo- porters may exist in an open-inwards conformation when nucleotide globin synthesis still occurs in the absence of ABCB10. Another is bound. We discuss ways in which this observation can be aligned role proposed for ABCB10 or potential homologs is stabilization with the current views on mechanisms of ABC transporters. of the iron transporter mitoferrin-1 (SLC25A37) (17, 18). A study of the yeast homolog of ABCB10 multidrug resistance-like 1 ABC mitochondrial erythroid | X-ray crystallography | (41% identity) (19) suggested that the substrates of ABCB10 may human membrane protein structure | nucleotide complex | cardiolipin be peptides of 6–20 amino acids that result from digestion of proteins by the m-AAA protease in the mitochondrial matrix TP-binding cassette (ABC) transporters move small mole- (20). To improve our understanding of ABCB10 function and Acules, ions, hormones, lipids, and drugs across cell membranes, to facilitate the identification of substrates, we have solved the and have diversified to act as ion channels and components of crystal structure of human ABCB10 in complex with nucleotide multiprotein complexes (1, 2). ABC transporters have critical roles analogs and in a ligand-free, apo form. These structures have given in many diseases, including juvenile diabetes (3), cystic fibrosis (4), and drug resistance in cancer (5). ABC transporters are ubiquitous proteins, bacteria having several hundred examples and humans 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., having 48 homologs (6). These diverse proteins share a common 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., architecture with two nucleotide binding domains (NBDs) and two 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., transmembrane domains (TMDs). The NBDs bind and hydrolyze and E.P.C. analyzed data; and C.A.S., A.C.W.P., and E.P.C. wrote the paper. fl ATP, providing the energy to move substrates across membranes The authors declare no con ict of interest. against a concentration gradient. The TMDs are more diverse, with *This Direct Submission article had a prearranged editor. several possible folds in bacteria, which provide binding sites for Freely available online through the PNAS open access option. a broad range of substrates (reviewed in refs. 1 and 2). ABC trans- Data deposition: The atomic coordinates and structure factors have been deposited in the porters function by an alternating access mechanism, where the Protein 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)]. TMD substrate binding sites alternate between outward- and in- 1C.A.S. and A.C.W.P. contributed equally to this work. ward-facing conformations (7). Structures of ABC transporters with 2Present address: Section of Structural Biology, Institute of Cancer Research, Chester the exporter fold have been obtained without bound nucleotide in Beatty Laboratories, London SW3 6JB, United Kingdom. the open-inwards conformation (8–10) and with nucleotides bound 3Present address: Institute of Molecular Biology and Biophysics, Eidgenössiche Technische in the open-outwards conformation (9, 11). However, the role of Hochschule Zürich, 8093 Zürich, Switzerland. conformational changes, the mechanism by which ATP hydrolysis 4Present address: Department of Human and Health Sciences, School of Life Sciences, drives transport and the sequence of substrate and nucleotide University of Westminster, London W1W 6UW, United Kingdom. binding remain controversial (2). 5To whom correspondence should be addressed. E-mail: [email protected]. ABCB10 (also known as ABC mitochondrial erythroid, ABC- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. me, mABC2, or ABCBA) is one of the three ABC transporters 1073/pnas.1217042110/-/DCSupplemental.

9710–9715 | PNAS | June 11, 2013 | vol. 110 | no. 24 www.pnas.org/cgi/doi/10.1073/pnas.1217042110 Downloaded by guest on September 30, 2021 (DDM) with the addition of either cholesteryl-hemisuccinate (CHS) or the mitochondrial lipid cardiolipin (CDL). We obtained crystals of ABCB10 both without nucleotide analogs and in the presence of the 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, which were phased by isomorphous replacement using a single mercury derivative. We subsequently obtained rod-form crystals that were solved by molecular replacement. The structure solution methods are summarized in Materials and Methods and are described in detail in SI Materials and Methods. Crystallographic statistics are available in Table S1 and the quality of maps is shown in Fig. S1. The overall fold of ABCB10 (Fig. 1A)iscommontomosteu- karyotic ABC transporters and some bacterial exporters, and was previously observed for the bacterial multidrug transporter Sav1866 Fig. 1. Structure of ABCB10 in complex with the nonhydrolysable nucleo- (11), the lipid flippase MsbA (9), and the mouse multidrug efflux tide analog AMPPCP, showing that ABCB10 is in an open conformation, even protein P-glycoprotein (mP-gp) (8) and its Caenorhabditis elegans when it is bound to nucleotide analogs. Cartoon representations of the homolog (ceP-gp) (10). The ABCB10 fold consists of a short ABCB10/AMPPCP complex monomer (A)andhomodimer(B)asseenintherod- α form B crystal structure. The structures have a single monomer in the asym- N-terminal -helix, lying parallel to the plane of the membrane, metric unit, the dimer is generated by a crystallographic twofold. followed by six long transmembrane α-helices (TMH) that tra- verse the lipid bilayer and project a further 30 Å into the mitochondrial matrix. The two monomers are interconnected by a us an unexpected conformation for nucleotide analog complexes domain swap where TMH4 and TMH5 interact with TMH1–3 and and new insights into the transport cycle for ABC transporters of TMH6 from the other half of the dimer (Fig. 1B). The TMD is the exporter fold. connected to the C-terminal NBD by an extended linker. The C- terminal domain adopts a classic NBD fold for an ABC transporter, Results with a RecA-like core subdomain, an α-helical subdomain, and Structure ABCB10 in Complex With Nucleotide Analogs and Without a disordered C terminus. ABCB10 is a homodimeric half trans- Bound Substrate. To solve the structure of ABCB10, we expressed porter and, in both the crystal forms, there is one monomer in the the protein in insect cells without the N-terminal mitochondrial asymmetric unit. The dimer is generated by a crystallographic targeting sequence as this improved protein yields. Two constructs twofold axis, so the two halves of the dimer form a symmetrical of ABCB10 were purified in the detergent dodecyl maltoside structure (Fig. 1B). BIOCHEMISTRY

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 in the open-inwards conformation [PDB ID code: 4F4C (10)] and Sav1866 [PDB ID code 2HYD (11)] in the open-outwards conformation. Transporters are viewed perpendicular to their (pseudo) twofold symmetry axes. (B) Alignment of NBDs of the structures shown in A. viewed looking toward the membrane. The ABCB10 monomers (blue/purple and orange/red respectively), nucleotides (green), and the NBD’s C-loop (cyan) are highlighted. The black lines/circles below each NBD pair indicate the translation required to bring the NBDs in the closed conformation for catalysis [the distance is the separation between the nucleotide γ-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 for proper 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 by superposition of the AMPPCP complex. The C-terminal extension in the Sav1866 structure has been omitted for clarity.

Shintre et al. PNAS | June 11, 2013 | vol. 110 | no. 24 | 9711 Downloaded by guest on September 30, 2021 Although ABC transporters of the exporter family have the same overall fold, they adopt radically different conformations during the transport cycle as they move from open-inwards to an open-outwards state, allowing the transporters to bind substrate on one side of the membrane and release the substrate on the other side. In the absence of nucleotide analogs, an open-inwards conformation has been observed where the NBDs are not in contact and the molecule forms an extensive substrate binding cavity facing the NBDs. This is the conformation we observe for our nucleotide-free ABCB10 structure (Fig. 2A) and was previously observed for MsbA (9), mP-gp (8), and ceP-gp (10) (Fig. 2A). The alternate open-outward conformation was found for complexes of ADP, ADP/vanadate, and AMPPNP with Sav1866 (11) (Fig. 2A) and MsbA (9). In the latter conformation, the NBDs are closely packed with two nucleotides sandwiched between the NBDs at the interface between the RecA-like core subdomain of one NBD and the α-helical subdomain of the second (Fig. 2B). In addition, a heterodimeric ABC transporter, TM287/288, from the thermophilic bacterium Thermotoga maritima has been solved in an intermediate open-inwards conformation with AMPPNP bound only to the noncanonical, high ATP affinity site, not to the catalytic site. In this case the NBDs are in contact at the high-affinity ATP binding site, but not at the catalytic site (21). Fig. 3. Interactions of nucleotides with the ABCB10 inward-facing confor- Unexpectedly, ABCB10 in complex with nucleotide analogs mation. (A) Schematic representation of NBDs of ABCB10 homodimer in the is in an inward-facing conformation with the NBDs separated, highest-resolution structure (rod form A) viewed looking from the TMD. AMPPCP (green/orange) are bound to each NBD but do not make inter-NBD similar to the conformation without bound nucleotide (Fig. 2A). interaction with the ABC transporter consensus sequence LSGGQ C-loop motif We observe clear density for both nucleotide and the magnesium (cyan). (B) Individual NBD viewed looking from adjacent NBD. NBD (pink) with ion in the higher-resolution rod-form crystals with bound AMPPCP. ABC Transporter nucleotide binding signature motifs colored as in C.TMD In the lower resolution plate-form crystals with AMPPNP, the (gray) and coupling helices (CH1, orange; CH2#, purple) are highlighted. Omit − 2+ σ NBDs are more disordered and have higher B factors, and here Fo Fc density for AMPPCP/Mg (blue mesh) is shown contoured at 3 . the density is only clear for the base, ribose ring and two phos- Dotted box indicates zoomed region in C and D. Detailed view of nucleotide phates (Fig. S1 D–F). The open-inwards conformation is observed binding site in rod form A/AMPPCP complex (C) and nucleotide-free form (D). in two crystal forms with unrelated packing (Fig. S2) and in sev- Oxygen atoms are colored red, nitrogen atoms blue, phosphate atoms orange eral crystals for each crystal form. There is clearly considerable and carbon atoms are colored according to the location of the atom: The fl AMPPCP carbon atoms are shown in green. The conserved NBD sequence exibility in the structure, different crystals giving structures with motifs are colored Walker A (yellow), Walker B (light blue), A-loop (pale cyan), variation in the extent of opening or closing of the open con- Gln-loop (red), His-loop (light green), coupling helix 1 (CH1, orange), and formation (Fig. 2), (Fig. S3 and Note 1 in the SI Materials and coupling helix 2 (CH2, magenta). The C-loop (cyan) is not visible as it is more Methods provide further information on the differences between than 16 Å away from the nucleotide binding site in this conformation. Resi- open conformation ABCB10 structures). However, in no case did dues/secondary structure elements marked with a pound symbol (#) denote we observe a change to the open-outwards conformation seen in regions contributed by homodimer partner. The side-chain of His690 is disor- MsbA and Sav1866 or the half-closed NBDs seen the TM287/288 dered and has not been modeled in rod form A. structure (Fig. S4). The flexibility we observe for the ABCB10 structures is in line with that observed for other ABC transport- the interactions observed for isolated monomeric NBDs with ADP ers in the open conformation, such as mP-gp (8) and MsbA (9). (22), bacterial maltose importer with AMPPNP, and maltose (23) Molecular dynamics simulations on the two high-resolution nucleotide complex structures of ABCB10, embedded in a phospho- and one site of TM287/288 (21). In both nucleotide-bound and lipid bilayer, indicated that overall the structure is stable during a nucleotide-free forms of ABCB10 the Walker B motif glutamate – (Glu659) adopts an unusual position, rotated 180° away from its 100-ns simulation (Fig. S5 A C), the only change in the structure γ occurring around TMH6, which has a tendency to unwind between expected orientation adjacent to the -phosphate. This conformation Gly446 and Gly447 at the glycine-rich sequence G458LGAGG in has been observed in isolated monomeric NBDs for ABCB6 (22). all four simulations (Fig. S5D). The His-loop histidine (His690) side-chain is partially disordered (not modeled in the rod-form A structure) and is further away γ ABCB10 Nucleotide Binding Site. The nucleotide binding site in from the -phosphate site than is usually observed in ABC ABCB10 is only partially formed. In earlier ABC transporter nu- transporters. We would expect these residues to move to the more cleotide complexes in the open-outwards conformation, the NBDs commonly observed positions once the NBDs come together. are tightly packed together, with two nucleotides sandwiched at the In the absence of nucleotide the ABCB10 NBDs have a similar interface, each interacting with both NBDs. However, in the conformation, except in the region of the Walker A motif, resi- ABCB10/AMPPCP structure the NBDs are separated to varying dues 530–534 (Fig. 3D), which form an additional turn of the degrees (Fig. 2B) and each NBD interacts with a single nucleotide Walker A helix when there is no nucleotide bound. In the nu- (Fig. 3A). ABC transporters have highly conserved nucleotide cleotide complexes these residues form an extended loop struc- binding sites with a series of conserved motifs that interact with the ture that interacts with the β/γ-phosphates of bound nucleotide nucleotide. The interactions between the nucleotide and the (Fig. 3C). This change in the local conformation of the nucleo- Walker A motif, A-loop tyrosine (Tyr501), and the Q-loop gluta- tide binding site is observed in some (22) but not all (8) nucle- mine (Gln575) from one NBD are very similar to those in other otide-free ABC transporter NBDs. ABC transporter structures (Fig. 3C), but the interaction with the The classic ATP switch mechanism suggests that when two ABC transporter consensus LSGGQ sequence (C-loop) from nucleotides bind to an ABC transporter dimer, it should convert to the other NBD is absent (Fig. 3A). This finding is reminiscent of the open-outwards conformation (24), but if only one of the two

9712 | www.pnas.org/cgi/doi/10.1073/pnas.1217042110 Shintre et al. Downloaded by guest on September 30, 2021 nucleotide binding sites has nucleotide bound, then the structure would remain in the open-inwards conformation. If this hypothesis were true, then for our homodimeric structures with a single monomer in the asymmetric unit, we would expect to see an oc- cupancy for the nucleotide of 50% or less. However, this result is not what we observe; refinement of the highest-resolution ABCB10 structure with fixed occupancies for the nucleotide and the magnesium ion of either 50%, 80%, or 100%, gave B factors for the nucleotide that were 30 Å2 below, similar to, or 30 Å2 above the average B factor for residues adjacent to the bound + nucleotide. The occupancies of the nucleotide and Mg2 are therefore in the 80–100% range, confirming that the majority of Fig. 4. ABCB10 has cardiolipin and detergent bound to the transmembrane dimers would have two nucleotides bound. This structure, there- helices and a portal between helices TMH1 and TMH2, which is open in the rod crystal form and closed in the plate-form crystals. (A)OverviewoftheABCB10 fore, represents an open-inwards conformation with nucleotide structure showing the location of lipid (magenta) and detergent (green) bound to both nucleotide binding sites in the majority of molecules. binding sites. (B) Molecular surface representation of the TMD in rod form A crystals, with lipid and detergent molecules shown in magenta and green and ABCB10 ATPase Activity and Inhibition by Nucleotide Analogs. Be- the portal between TMH1 and TMH2 indicated with a dotted line. In the rod- cause the open-inwards, nucleotide-bound conformations are un- form structures TMH1 and TMH2 are loosely packed revealing a 7 Å wide × 30 Å expected, we investigated whether the ABCB10 ATPase activity long portal connecting the central cavity of the TMDs with the membrane and inhibition of this activity by nucleotide analogs differed from environment. The portal is occupied by a CDL alkyl chain (magenta). (C)Inthe those of other ABC transporters. ABC transporters use the binding plate form crystals TMH1 and TMH2 are packed closer together, with the portal closed. Structures are viewed in the same orientation as in A. and hydrolysis of ATP to power the movement of substrates. Sur- prisingly, many ABC transporters exhibit a basal ATPase activity in vitro in the absence of transport substrate, which is stimulated be- nucleotide analogs is therefore of the same order-of-magnitude – tween 2- and 10-fold when a transport substrate is added (25 27). as its affinity for ATP, suggesting that the interactions observed ABCB10 showed basal ATPase activity, with apparent kinetic for the analogs would be similar to those for ATP. parameters similar to those of other ABC transporters (28, 29), when it was purified in DDM and either CHS or CDL, with or Portal Between Two Transmembrane Helices Could Be a Route for without reconstitution into liposomes (Table 1, Fig. S6 A and B,and Substrate Entry. Lipid and detergent molecules are clearly visible SI Materials and Methods). It is clear that ABCB10 is active both in interacting with the outer surface of the membrane-spanning re- micelles and in proteoliposomes (Fig. S6 A and B). Small increases gion in all four ABCB10 structures (Fig. S7). Intriguingly in the in Kcat/Km, of the order of fourfold on reconstitution are a common rod-form crystals, TMH1 and TMH2 are separated, with elon- feature of many ABC transporters (29–31). The basal ATPase ac- gated electron density between these helices, which we have at- tivity is inhibited by vanadate (Fig. S6 A and B). CDL does not tributed to an alkyl chain of a CDL molecule (Fig. 4 A and B). This stimulate the ATPase activity when added during reconstitution lipid chain contacts both the internal cavity and the outer surface (Fig. S6C), suggesting that it is unlikely that CDL is transported by of the protein. The portal through which this CDL passes links the ABCB10. Mutation of the conserved putative catalytic Glu659 to internal TMD cavity and the center of the lipid bilayer. In contrast, glutamine in the nucleotide binding site of ABCB10 gave protein in the plate-form crystals these helices are packed tightly together, with no detectable activity (Fig. S6 B and E), as is observed for other with no opening between them (Fig. 4C). In the rod-form crystals ABC transporters (32, 33). The first construct used for crystalliza- there are three residues which form crystal contacts to the loop tion had a PCR-derived mutation, which converted Arg691 to joining TMH1 and TMH2, but there are no crystal contacts in the a histidine. The R691H mutation led to a substantial loss of activity transmembrane region involving these helices, so it is unlikely that and ATPase activity was restored when the Arg691 was reintro- this change in conformation is induced by the crystal contacts duced (Fig. S6D). The significance of this observation is discussed in observed in the rod-form crystals. This portal could provide Note 3 of SI Materials and Methods. a route of entry for a hydrophobic or amphipathic substrate from The nucleotide analog complex structures presented here have the membrane into the binding cavity. AMPPNP or AMPPCP bound to the NBDs. We investigated the inhibition of the ABCB10 ATPase activity by these nucleotide Conserved Residues in the TMD Suggest a Substrate Binding Site for analogs (Fig. S6F). AMPPCP and AMPPNP have IC50s of 2.3 ± ABCB10. Alignment of the protein sequences of ABCB10 paralo- 0.9 mM and 1.1 ± 1.1 mM, respectively, measured with 2 mM gues in 80 organisms highlights conserved patches that are ex- BIOCHEMISTRY ATP. Using the Km,app of 0.2 mM measured for this protein posed on the inner surface of the transporter (Fig. 5 and Fig. S8). sample, the apparent Ki for AMPPCP is of the order of 0.2 mM The external surface of ABCB10 is not conserved, suggesting and for AMPPNP is 0.1 mM. The affinity of ABCB10 for these that ABCB10 has a role in substrate transport rather than forming a complex with another protein, which would require a conserved external surface. The NBD signature motifs are highly conserved, Table 1. Kinetic parameters for the basal ATPase activity together with a patch formed by residues at the N-terminal end of ABCB10 of TMH3 and the C-terminal end of TMH6. This latter cluster

Km, app Vmax, app interacts closely with conserved residues in the domain-swapped † ‡ −1 −1 -1 -1 Lipid* Recon n (ATP) mM (nmol Pi min ·mg ) Kcat/KmM ·s TMH4 (Q344/A348) in the open-out conformation. A third cluster is located on the concave surface formed by the TMD. There are CHS − 2 0.18 ± 0.02 51 ± 1.1 2.2 two conserved motifs, (N/I)xxR located in the center of TMH2 and CHS + 10 0.3 ± 0.08 319 ± 27 8.2 NxxDGxR at the N terminus of TMH3B, which form a patch on the CDL − 2 0.5 ± 0.2 106 ± 18 1.6 inner surface of the TMD cavity (Fig. 5 B–D). These residues form CDL + 3 0.19 ± 0.03 181 ± 9.8 6.7 an ABCB10 signature sequence and, based on their location, we *Protein puriﬁed in DDM, with the addition of either CHS or CDL. suggest that they could form part of the substrate binding site for † Protein in detergent (−) or reconstituted into proteoliposomes (+). an amphipathic substrate. If the outward-facing conformation of ‡n = number of independent puriﬁcations. ABCB10 resembles that of Sav1866 (11), then the two helices

Shintre et al. PNAS | June 11, 2013 | vol. 110 | no. 24 | 9713 Downloaded by guest on September 30, 2021 Fig. 5. Sequence conservation of residues in ABCB10. Conservation is mapped onto a molecular surface representation of the concave inner cavity (A) and convex outer surface (B) of ABCB10. The molecular 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 second monomer (residues 311–424). Residues are colored according to sequence conservation, invariant (dark blue), highly conserved (slate; 2–4 related amino acids), and moderately conserved (pale blue; 3–8 related amino acids). Sequence conservation was calculated based on an alignment of eighty ABCB10 homologs (human to yeast) using the CONSURF server (38). The conserved patch in the TMD between 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 semitransparent molecular surface. (E) Conserved signature sequence defined by residues located in central portion of TMH2 and at the N-terminal end of TMH3B.

TMH2 and TMH3 would be substantially further apart in the the nucleotide binding before the substrate, was suggested for the open-outward structure, changing the substrate binding site and transporter associated with antigen processing (34) and alternative therefore releasing the substrate into the intermembrane space. binding sequences have also been proposed for P-gp (35). Alter- nating catalytic site mechanisms involving asymmetric binding and Discussion hydrolysis have also been put forward (reviewed in ref. 36). ABCB10 Structure and ABC Transporter Mechanism. Our four struc- Our structures suggest an adaptation of the accepted ATP tures of ABCB10 in complex with nucleotide analogs and without switch mechanism in which either transport substrate or nucle- bound nucleotide give snapshots of ABCB10 before substrate otide could bind first (Fig. 6). These ABCB10 nucleotide analog binding and in a conformation where ATP has bound, and the complex structures show that nucleotides can bind in the absence protein is awaiting the transport substrate. ABCB10 remains an of a transport substrate, an observation that is in line with the orphan transporter, one for which we do not yet know the transport fact that many ABC transporters, including ABCB10, have a basal ATPase activity, performing the ATPase reaction in the absence substrates. Our structures do, however, suggest a potential entry of transport substrate. ABC transporter NBDs must therefore route for an amphipathic substrate and a potential binding site for be capable of binding ATP and coming together in a productive transport substrates. complex for ATP hydrolysis, even when there is no transport The generally accepted ATP switch model for ABC transporter substrate present. function (24) proposes that binding of nucleotide to the protein/ Although our structures indicate that ABC transporters can substrate complex is the trigger for conversion from the open-in- bind nucleotide in the absence of transport substrate, it has also wards to the open-outward conformation, thus driving transport of been shown that many ABC transporters bind transport sub- the substrate across the membrane. A similar mechanism, but with strate and inhibitors in the absence of nucleotide, as has been observed for P-gp (8) and transporter associated with antigen processing (37). We therefore propose that ABC transporters could bind either nucleotides or transport substrate first, each Substrate 2 ATP binding initially to one face of open-inwards conformation. Once # the nucleotides or transport substrate binds to one face of the complex, the NBDs or TMDs could come together in a productive orientation, forming complete binding sites with interactions for the substrate molecules with both halves of the TMDs and for the ATPs with both NBDs (Fig. 6). Because the rate of ATP turnover is often higher in the presence of the transport substrate, occupation of the transport substrate binding site must promote 2 ATP 2 ADP View formation of the ATP hydrolysis-competent closed NBD con- 90° Substrate rotated 90° formation. The NBDs coming together in the presence of bound transport substrate would then trigger reorganization of the TMDs into the open-outwards conformation, disrupting the transport substrate binding site and forcing the substrate to dissociate on the other side of the membrane. One or both of the nucleotides would then be hydrolyzed and dissociate, allowing 2 Pi the structure to return to the inward-facing conformation. Al- though our structures are symmetrical, having a monomer in the asymmetric unit, this does not preclude the possibility that cer- tain steps in the mechanism involve asymmetry in the nucleotide binding site, with hydrolysis occurring in only one site, as has Fig. 6. Overview of the steps proposed for the transport cycle of ABCB10 been shown for other ABC transporters (36). These ABCB10 and other ABC transporters of the exporter family. The TMDs are colored blue and orange, with their associated NBDs colored purple and red. ATP is structures therefore provide fresh insights into ABC transporter shown in green, ADP in gray and the transport substrate in yellow. An as- function, in particular providing a unique example of exporter terisk (*) and pound symbol (#) indicate the conformations observed for fold ABC transporters in complex with nucleotide analogs, in an nucleotide-bound and nucleotide-free ABCB10, respectively. open-inwards conformation. The findings suggest that nucleotide

9714 | www.pnas.org/cgi/doi/10.1073/pnas.1217042110 Shintre et al. Downloaded by guest on September 30, 2021 binding can occur before transport substrate binding and that it functional studies we reconstituted ABCB10 into liposomes containing the is therefore likely that the order in which these molecules bind synthetic lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and is not fixed. Binding of the transport substrate does, however, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine (POPE) without the promote the formation of the conformation in which the NBDs addition of CHS or CDL. come together, converting the TMDs to the open-outwards con- We obtained plate-form crystals with both ABCB10 constructs, purified in formation, thus leading to a stimulation of the ATPase reaction DDM and CHS, without addition of nucleotide and with the nucleotide analogs and transport of the substrate. AMPPNP or AMPPCP. All nucleotide analog stocks were prepared with equi- The key role of ABCB10 in erythropoiesis and relief of oxi- molar magnesium chloride. Crystals were grown at 20 °C using the sitting-drop dative stress suggests that this transporter could be an interesting vapor-diffusion method. Diffraction data were collected on beamline I24 at candidate to explain a frequently observed adverse effect of Diamond Light Source. Crystals grown with AMPPNP diffracted anisotropically drugs and early clinical inhibitors in red blood cell formation, beyond 3.3 Å and were used to solve the structure with phases from a single resulting in anemia or decreased recovery of cardiac function mercury derivative (Fig. S1 and Table S1). The chain trace was confirmed with after ischemia/reperfusion. The future identification of ABCB10 data from selenomethionine-labeled ABCB10 crystals. Rod-shaped crystals substrates and binding studies of inhibitors and drugs that have were obtained without nucleotide and with AMPPCP from protein purified in been associated with these side effects will facilitate identifica- the presence of CDL. The rod-form crystals diffracted to at least 2.9 Å and tion of the function of ABCB10. Such studies would also give were phased by molecular replacement using an initial model derived from insight into the role of ABCB10 in conditions causing increased the plate form. ATPase activity assays and molecular dynamics simulations mitochondrial oxidative stress, such as aging, anemia, cardiac methods are described in SI Materials and Methods. ischemia/reperfusion, or neurodegenerative diseases and its po- ACKNOWLEDGMENTS. We thank Robert Tampé and Peter Henderson for tential as a target for pharmaceutical intervention. helpful discussions and Stefan Knapp for critical reading of the manuscript; Richard Callaghan for help with establishing the ATPase assay; Tobias Krojer, Materials and Methods Melanie Vollmar, and Joao Muniz for assistance with crystal screening; the The complete methods are presented in SI Materials and Methods.For staff at Diamond Light Source and, in particular, the microfocus beamline structure and function studies we expressed ABCB10 in insect cells using I24 for assistance with crystal screening and data collection. The Structural Genomics Consortium is a registered charity (no. 1097737) that receives baculovirus vectors. We expressed ABCB10 with both N- and C-terminal His- funds from the Canadian Institutes for Health Research, Genome Canada, tags with the mitochondrial targeting presequence (mTP) removed, either GlaxoSmithKline, Lilly Canada, the Novartis Research Foundation, Pfizer, – by deletion of the N-terminal 151 residues or removal of residues 6 126. We Takeda, AbbVie, the Canada Foundation for Innovation, the Ontario Min- extracted and purified ABCB10 in the detergent DDM with the addition of istry of Economic Development and Innovation, and the Wellcome either CHS or CDL by cobalt affinity and size-exclusion chromatography. For Trust (092809/Z/10/Z).

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