Structural insights into toxicant export mediated by ABCC2 in Arabidopsis thaliana

Domain: biology
Published: 2025-12-22
Editorial outcome: major_revision

Abstract
Plants are highly vulnerable to damage from environmental pollutants, making detoxification mechanisms essential for sustaining growth and development. ABCC2 in Arabidopsis thaliana (AtABCC2) plays a critical role in detoxification by exporting diverse toxic compounds. Here, we report the structures of AtABCC2 in three distinct states: substrate-free, bound to the substrate S-(2,4-dinitrophenyl)glutathione (DNP-GS), and bound to ATP. Both monomeric and dimeric forms of AtABCC2 are observed. Unlike other dimeric ABCC homologs, AtABCC2 features a dimer interface mediated by its transmembrane domains. DNP-GS occupies an amphipathic cavity formed by the transmembrane domains. ATP binding drives the conformational changes in each protomer which bring the transmembrane and nucleotide-binding domains closer together, transitioning the transporter from a cytosolic-facing to an occluded state. Together, these findings advance our understanding of the molecular basis of substrate binding and transport by AtABCC2, and shed light on plant detoxification mechanisms.

Introduction
Plant cells are highly vulnerable to various natural or biosynthetic toxic substances, such as heavy metals and xenobiotics, and have evolved different types of detoxification mechanisms to ensure their survival and normal growth1,2. Isolating the toxicants in cellular compartments, particularly the vacuoles, can minimize their toxic effects in the cytosol and protect the cells from damage3,4. Typically, the toxicants are conjugated to molecules such as glutathione sulfate, amino acids, or chelators like phytochelatins or metallothioneins, after which they are transported into the vacuoles or expelled from the cytosol1,3,5,6. The ATP-binding cassette subfamily C (ABCC) transporters play a crucial role in the detoxification process by mediating the export of the toxicants or their conjugates7,8. In Arabidopsis thaliana, there are 15 members of the ABCC subfamily9. Arabidopsis thaliana ABCC1 (AtABCC1) and ABCC2 (AtABCC2) were identified as detoxification transporters located on the vacuole membrane10,11. They are widely expressed across various plant tissues, including roots, stems, leaves, flowers, and buds12. Notably, AtABCC2 exhibits a significantly higher transport capacity compared to AtABCC111. AtABCC2 is known for its broad substrate specificity and is capable of mediating the transport of diverse compounds, including heavy metals, herbicides, and pesticides13,14. It facilitates the transport of glutathione conjugates or glucuronic acid conjugates, including heavy metals like cadmium-glutathione conjugates, the glutathione conjugate S-(2,4-dinitrophenyl)glutathione (DNP-GS), and the glucuronic acid conjugate 17β-estradiol-(β-D-glucuronide)(E 17βG)15. DNP-GS is derived from 1-chloro-2,4-dinitrobenzene (CDNB), a compound which is predominantly used in the production of pesticides and exhibits a high cytotoxicity to plant cells16,17. Once absorbed into the cytosol by the plant cell, CDNB is conjugated to glutathione by the glutathione S-transferase (GST), and then exported into the vacuole by AtABCC215,17. In addition to exogenous toxic substances, AtABCC2 also plays a role in transporting various endogenous metabolites, such as chlorophyll metabolites18, β-D-glucopyranosyl abscisate (ABA-GE), oxidized glutathione (GSSG), and anthocyanins19–21.

The ABCC transporters, also known as multidrug resistance proteins (MRPs), are a crucial group of membrane transporters utilizing the energy of ATP hydrolysis to transport substrates, and are conserved across various species, including yeasts, plants, and mammals12,22. Topologically, the ABCC subfamily is classified into “long” and “short” transporters based on the presence or absence of an additional transmembrane domain (TMD0) located before the two core TMDs forming the substrate-binding cavity23,24. The TMD0 is connected to the core transmembrane domain through a long peptide, known as the lasso motif or L0 linker9. Previous studies have shown that the N-terminal extension of ABCC transporters, consisting of TMD0 plus L0 linker (or L0 linker alone), plays a crucial role in the proper localization of the protein, its interactions with other regulatory factors, and the regulation of its transport activity25–33. Structures of the ABCC subfamily transporters in Saccharomyces cerevisiae and mammals have been reported34–42. However, the protein sequences vary across species, particularly in the TMD0 domain (Supplementary Figs. 1 and 2a, b). The mechanisms by which plant ABCC transporters export toxicants remain unclear.

In this study, we characterize the ATPase activity of AtABCC2 under different compounds, and determine its structures using cryo-EM single-particle analysis under three distinct states: the substrate-free (apo) state, the substrate DNP-GS-bound state, and the ATP-bound state. Together, the structural and biochemical analysis of AtABCC2 provides insights into the mechanisms of substrate binding and transport, as well as the plant detoxification process.

Results
ATPase activity characterization of AtABCC2
We overexpressed the full-length wild-type (WT) AtABCC2 (AtABCC2 ) and a catalytic mutant, in which the conserved catalytic residue E1404 in the Walker B motif of the nucleotide-binding domain 2 (NBD2) was substituted by glutamine (designated AtABCC2 or AtABCC2 ), in human embryonic kidney (HEK) 293F cells. The proteins were purified with the detergent digitonin. Under this condition, only one peak corresponding to the AtABCC2 or AtABCC2 protein was observed in size-exclusion chromatography (Supplementary Fig. 3a, b). The peak fractions were collected and subjected to further biochemical and structural analysis (Supplementary Fig. 3a, b). We first examined the ATPase activity of AtABCC2 in the presence of varying ATP concentrations. The ATPase activity of AtABCC2 increased with rising ATP concentrations. The K and V values were determined to be 0.38±0.04 mM and 2,547±73 nmol∙min−1∙mg−1 protein, respectively, using the Michaelis–Menten equation (Fig. 1a). In contrast, the ATPase activity of the AtABCC2 mutant was significantly reduced compared to that of the AtABCC2 (Fig. 1a, b). We then tested the ATPase activity of the AtABCC2 in the presence of serial concentrations of different compounds which have been previously identified as substrates, including DNP-GS, E 17βG, PC , GSSG, GSH, and ABA-GE (Fig. 1c and Supplementary Fig. 3c). The results showed that the addition of DNP-GS, E 17βG, or PC significantly stimulated the ATPase activity of AtABCC2 in a concentration-dependent manner (Fig. 1c), with a maximum increase of about 60%, 53%, or 12%, respectively, as indicated by the fitted V values using the Hill equation (Fig. 1d, e). In contrast, GSSG, GSH, or ABA-GE induced only a modest increase in the ATPase activity, with a maximum increase of about 5% relative to the basal activity observed in the absence of the compounds (Fig. 1d, e). The fitted EC values for DNP-GS, E 17βG, and PC were 86.79±28.13 μM, 78.91±40.09 μM, and 2.42±1.46 μM, respectively (Fig. 1d). Additionally, the presence of DNP-GS or E 17βG resulted in an increase in V and a decrease in K for AtABCC2. The fitted K value in the presence of DNP-GS or E 17βG was 0.15±0.03 mM and 0.18±0.04 mM, respectively (Fig. 1f). These findings were consistent with previous reports, suggesting that AtABCC2 efficiently transports DNP-GS or E 17βG11,15.

Overall structure of the wild-type AtABCC2 in the apo state
With the purified protein in a detergent micelle, we determined the structure of AtABCC2 in the apo state using the cryo-EM single-particle analysis method (Supplementary Figs. 4a–h and 5a). Notably, during particle 2D and 3D classifications of the data processing steps, protein particles in both the monomeric and the dimeric form were observed, and reconstructed separately (Supplementary Fig. 4b–h). The monomeric form was the predominant state, taking up about 47% of the selected particles, while the dimeric form accounted for about 26% (Supplementary Fig. 5a). Finally, we obtained the EM density maps of AtABCC2 in both the monomeric and the dimeric forms, with overall resolutions of 3.8 Å and 4.2 Å, respectively (Fig. 2a and Supplementary Figs. 4c, f, and 5a). The local resolution of the monomeric form was better for the TMDs of AtABCC2 than for the NBDs, enabling side chain assignment for the TMDs (Supplementary Figs. 4e, h, and 5b, c). Notably, the local resolution of the TMDs on the vacuolar side was lower than the rest, suggesting intrinsic flexibilities of this region. The structure model was built combining both manual building and the predicted model from AlphaFold243. As is typical for ABC transporters, the core structure of AtABCC2 consists of two TMDs (TMD1 and TMD2) and two NBDs (NBD1 and NBD2) (Fig. 2b, c). TMD1 is formed by transmembrane segments (TMs) TM6-TM11, whereas TMD2 consists of TM12-TM17 (Fig. 2b–d). The two TMDs are arranged in a typical domain-swapped manner of the type IV ABC transporter44: TM6-TM8, TM11, TM15, and TM16 form one structural unit (TM bundle 1, TMB1), whereas TM9, TM10, TM12-TM14, and TM17 form the other (TM bundle 2, TMB2). The two TMBs exhibit a “V”-shaped architecture, creating a large cavity facing the intracellular side (Fig. 2d, e). It suggests that the captured WT AtABCC2 structure in the absence of substrates represents an inward-facing state, which is typical for the ABC transporters adopting similar structures44–46.

Like the “long” ABCC homologs24, the structure of AtABCC2 consisted of an additional TMD0 and a linker (L0) preceding the first transmembrane domain, TMD1. TMD0 has five transmembrane helices (TM1 to TM5) (Fig. 2b, c). The L0 linker contains three helical structures, with the last helix oriented almost parallel to the membrane surface and positioned on the exterior region of TMB1, close to TM6, TM7, TM11 and TM16 (Fig. 2b). Structural comparison revealed that, although the L0 linker of AtABCC2 aligned well with the ABCC homologs represented by the human ABCC2 (hABCC2)42,47 and ABCC3 (hABCC3)41, TMD0 exhibited a significant lateral shift (Fig. 2f). In hABCC2 and hABCC3, TMD0 is positioned laterally relative to TMB1, whereas in AtABCC2, TMD0 is located between the two TMBs (Fig. 2f). Human ABCB6 (hABCB6), which is a half-transporter belonging to the ABCB subfamily, also contains a TMD0 domain48. The TMD0 domains in hABCB6 and AtABCC2 show different localizations relative to the core TMBs as well (Supplementary Fig. 6a). This suggests that the extra N-terminal TMD0 domain displays structural variations among the ABC transporters. Based on sequence alignment results, the TMD0 exhibits low sequence conservation, thus possibly leading to the varied orientations observed in the structures (Supplementary Fig. 1a). In AtABCC2, TM1 and TM4 of TMD0 interact with TM15 and TM16 of TMB1, primarily through hydrophobic packing and hydrogen bonding (Fig. 2g). To assess the functional roles of TMD0 and L0 linker, we generated two truncated versions of AtABCC2: one with the TMD0 truncated (ΔTMD0) and the other with both the TMD0 and L0 truncated (ΔTMD0+L0), and measured their ATPase activities. The truncated variants were well-folded as indicated by the results of the size-exclusion chromatography (Supplementary Fig. 6b). The ΔTMD0 mutant exhibited similar ATPase activity compared to the wild-type protein, with a fitted V value of 2824±73 nmol∙min−1∙mg−1 protein and a K value of 0.74±0.06 mM. In contrast, truncation of both the TMD0 and L0 linker resulted in a dramatic reduction in ATPase activity compared to the WT protein, with a fitted V value of 2737±354 nmol∙min−1∙mg−1 protein and a K value of 2.25±0.53 mM (Supplementary Fig. 6c, d). These results are consistent with previous studies, suggesting that while TMD0 has minimal impact on the transport and ATPase activity of ABCC transporters, the L0 linker is essential to substrate transport, likely by fine-tuning the conformational changes required for substrate translocation26,31,41.

The dimeric form of AtABCC2 exhibits a two-fold C2 symmetry (Supplementary Fig. 6e). The monomeric structure model can be docked well into the EM map in the dimeric state, supporting the same conformation for the monomeric protein (Supplementary Fig. 6f). The dimer interface of AtABCC2 is formed by both the TMB regions and the NBDs of the two protomers (Supplementary Fig. 6f). In the TMB region, the interface is mediated by hydrophobic interactions between TM12 and TM13 from each protomer (Supplementary Fig. 6f). In the NBD region, NBD2 from one protomer packs to NBD2 from the other protomer (Supplementary Fig. 6f). Due to the lower local resolutions of the NBDs in the EM map, we were unable to analyze the interactions in this region in more detail.

To confirm the observed monomeric and dimeric forms of AtABCC2 observed in the cryo-EM sample, we carried out the mass photometry (MP) analysis of the purified protein in the detergent of digitonin. Consistent with the EM analysis, the MP data showed that AtABCC2 exists in two major forms, the predominant one (approximately 67%) corresponding to the monomeric form with a molecular weight of 206 kDa which is close to the theoretical value of 182 kDa, and the other one (approximately 33%) corresponding to the dimeric form with a molecular weight of 407 kDa close to the theoretical value of 364 kDa (Supplementary Fig. 7a). Meanwhile, the ΔTMD0 mutant showed similar oligomeric patterns to the WT protein (Supplementary Fig. 7a), consistent with the structural observation that TMD0 is not involved in the dimerization of AtABCC2. To separate the two oligomeric species, we tried to purify the protein under other detergent conditions. In the detergent of lauryl maltose neopentyl glycol (LMNG) plus cholesteryl hemisuccinate (CHS), the AtABCC2 protein displayed two peaks in size-exclusion chromatography (Supplementary Fig. 7b, c). Fractions of each peak were collected and subjected to MP analysis. The first peak predominantly (approximately 83%) had a molecular weight of 425 kDa corresponding to the dimeric form (Supplementary Fig. 7d), while the second peak predominantly (approximately 80%) had a molecular weight of 248 kDa corresponding to the monomeric form (Supplementary Fig. 7e). We characterized the ATPase activity of the two species. The results showed that the ATPase activity of the monomeric and dimeric forms had no significant differences either in the absence or presence of the substrate DNP-GS (Supplementary Fig. 7f).

Structure of the AtABCC2 mutant in the apo state
We then determined the cryo-EM structure of AtABCC2 mutant in the apo state (Supplementary Fig. 8a–f). During data processing, we also observed particles in both the monomeric and dimeric forms (Supplementary Fig. 8f). However, in contrast to the AtABCC2 , the dimeric form was predominant (Supplementary Fig. 8f). The mass photometry results are consistent, showing a higher ratio of the dimer than the monomer in the AtABCC2 protein solution (Supplementary Fig. 7a). Due to the limited number of particles in the monomeric form, we were unable to reconstruct a high-resolution EM map (Supplementary Fig. 8f). The EM map of the dimeric form was reconstructed with a C2 symmetry. Finally, we obtained a map of the dimeric AtABCC2 at an overall resolution of 3.5 Å (Fig. 3a and Supplementary Fig. 8c–e). Compared to the EM map of the dimeric AtABCC2 , the local resolutions of the transmembrane region and the NBDs involved in the dimer interface were higher, allowing accurate residue assignment (Supplementary Fig. 8e, g). Like the AtABCC2 , the dimer interface of AtABCC2 was formed by TMB2 and NBD2 from both protomers (Fig. 3b, c). TM12 and TM13 from the two protomers mediated the dimer formation mainly through hydrophobic packing (Fig. 3c and Supplementary Fig. 9a). Compared with the dimeric AtABCC2 , TM12 and TM13 of each protomer in the dimeric AtABCC2 rotated about 10° (Supplementary Fig. 6g). Consequently, the two NBD2 domains of each protomer were closer than in the AtABCC2 structure, leading to a different NBD interface (Supplementary Fig. 6g). In the dimer interface of AtABCC2 , two clear EM densities corresponding to lipid molecules were identified, which can be docked well with phosphatidylcholines, each localized between TM14 from one protomer and TM13 from the other (Supplementary Fig. 10a). The hydrocarbon tails of the lipid molecule pack to the hydrophobic residues in TM13 and TM14 (Supplementary Fig. 10a). Thus, these lipid molecules contribute to the dimer formation of AtABCC2 by mediating additional contacts between the two protomers.

Structure alignment of the monomeric AtABCC2 structure and the protomer in the dimeric AtABCC2 structure revealed a root-mean-square deviation (RMSD) of 2.6 Å (1026 Cα atoms aligned) (Fig. 3d). While TMD0, L0 linker, TMB1, and NBD1 of the two protomers align well, TMB2 and NBD2 exhibit a rotational shift (Fig. 3d). Consequently, the two TMBs and NBDs are closer in the AtABCC2 structure compared to the AtABCC2 structure. While the vacuolar termini of the transmembrane segments in TMB2 remain unchanged, the intracellular parts of TMB2, including TM9, TM10, TM12-TM14, and TM17, shift toward TMB1, resulting in a more compact, intracellularly-facing cavity (Fig. 3d). This configuration of AtABCC2 may represent an intermediate state in the substrate transport cycle.

Recently, dimeric structures have also been reported for the yeast glutathione-conjugated heavy metal transporter Ycf1 and the human ABCC1 (hABCC1)39,40. Like AtABCC2, lipid or detergent molecules were identified to be involved in the dimer formation of Ycf1 and hABCC139,40. However, the dimer interface of Ycf1 or hABCC1 is formed mainly by the TMD0 of one protomer and the core TMDs of the other, distinct from that of AtABCC2 (Supplementary Fig. 10b). The buried surface area (BSA) analysis revealed that Ycf1 has the largest BSA value (approximately 1599 Å2) which is consistent with its more compact dimer organization observed in the structure, while AtABCC2 and hABCC1 have comparable BSA values (1058 Å2 and 1002 Å2, respectively) (Supplementary Fig. 10c–e).

DNP-GS binding site of AtABCC2
The ATPase activity analysis supports that DNP-GS conjugate is a transport substrate of AtABCC2. To investigate the toxicant recognition mechanism, we tried to get a DNP-GS-bound structure by adding DNP-GS to the purified AtABCC2 protein and subjecting the complex to cryo-EM analysis (Supplementary Fig. 11a–f). Like the apo AtABCC2 sample, particles in both the monomeric and dimeric forms were present (Supplementary Fig. 11e). We obtained EM maps of the dimer and monomer at an overall resolution of 3.5 Å and 4.1 Å, respectively (Supplementary Fig. 11b–e). The two maps can be superimposed well, suggesting almost identical structures (inset panel of Supplementary Fig. 11e). The EM density corresponding to one DNP-GS molecule was clearly identified within the transmembrane cavity (Fig. 4a, b). Electrostatic potential and hydrophobicsurface analysis revealed that the DNP-GS binding interface was amphipathic, with one region highly positive in electrostatic potential and another predominantly hydrophobic (Fig. 4c, d). Residues involved in DNP-GS binding were identified (Fig. 4e). Specifically, R1198 forms ionic interactions with the carboxyl group of the glutathione part of DNP-GS and the nitro group of the dinitrophenyl part of DNP-GS (Fig. 4e). The carboxyl group of the glutathione part is also close to the side chains of Q415, N1140, and R1141 (Fig. 4e). Additionally, the phenyl group of the dinitrophenyl part packs closely to the side chains of F1034, M1037, and L1199. F361 is also positioned closely to the glutathione part of DNP-GS (Fig. 4e). To confirm the roles of these residues in DNP-GS binding, we mutated each residue to alanine and tested the ATPase activity of the mutants in the presence of DNP-GS. The results showed that all mutants exhibited decreased ATPase activities compared to the wild-type AtABCC2, with the R1198A mutant displaying the most significant reduction (Fig. 4f). This suggests that both ionic and hydrophobic interactions are crucial for the binding of DNP-GS to AtABCC2, and both the glutathione and dinitrophenyl parts of DNP-GS are involved in the substrate recognition.

The DNP-GS-bound AtABCC2 structure adopts a similar inward-facing conformation to that of the apo-state AtABCC2 structure (Fig. 4g). Superimposing the two dimeric structures revealed an RMSD of 1.2 Å (1427 Cα atoms aligned). The dimer interface, formed by TMB2 and NBD2, remains unchanged in the two structures (Fig. 4g). Upon DNP-GS binding, the cytosolic parts of the TMs in TMB1 undergo a slight shift toward TMB2, resulting in a partial closure of the substrate-binding cavity. Consequently, NBD1 moves closer to NBD2 (Fig. 4h, i).

Substrate-bound structures have been determined for several ABCC homologs, including the E 17βG-bound hABCC3 structure, the LTC -bound bABCC1 structure, and the different ligand-bound human MRP2 and MRP4 structures34,37,41,47. Structural alignments of the DNP-GS-bound state of AtABCC2 , the LTC -bound bABCC1, and the E 17βG-bound hABCC3 structures reveal an RMSD of 2.4 Å (over 780 Cα atoms) and 3.4 Å (over 790 Cα atoms), respectively (Supplementary Fig. 12a). While hABCC3 binds two E 17βG molecules asymmetrically in its tapered cavity, both bABCC1 and AtABCC2 bind only one substrate molecule. In all cases, the substrate molecules bind to a highly conserved site within the corresponding transporters (Supplementary Fig. 12a, b). Notably, E 17βG, LTC , and DNP-GS are all amphiphilic molecules and fit well into the amphipathic cavity (Supplementary Fig. 12c). Comparison of the bABCC1 and AtABCC2 structures revealed highly conserved residues in this binding site, including the positively charged arginine residues and the aromatic residues (Supplementary Figs. 12b and 9c, d). The relatively large binding pocket could accommodate substrates of various sizes, which may contribute to the broad substrate specificity of the ABCC family transporters.

Conformational changes of AtABCC2 upon ATP binding
The binding of ATP to ABC transporters has been shown to facilitate the transport and subsequent release of substrates49. We added ATP to the purified AtABCC2 protein sample, and then performed cryo-EM analysis (Supplementary Fig. 13a–e). Similar to the apo-state and DNP-GS-bound samples, both monomeric and dimeric forms were present in the ATP-bound AtABCC2 particles, with the dimeric form being the predominant one (Supplementary Fig. 13e). Due to limited particle numbers, the EM map of the monomeric form was only determined at a low resolution (Supplementary Fig. 13e), precluding further analysis. Finally, we obtained an EM map of the ATP-bound dimeric structure at an overall resolution of 3.4 Å (Fig. 5a and Supplementary Fig. 13b–d). The local resolutions of both the TMDs and NBDs were significantly improved (Supplementary Fig. 13d, f), especially for the NBDs, suggesting that ATP binding stabilizes the NBDs. The density corresponding to the ATP molecule was clearly observed between the two NBDs (Supplementary Fig. 13f). Compared to the apo-state AtABCC2 structure, the dimer interface, as well as the TMD0 and L0 linker, remains unchanged (Fig. 5b, c). However, large conformational changes are observed between the two halves of the transporter. NBD1 and TMB1 move towards their counterparts (Fig. 5b–d). Structural analysis revealed that ATP binds to the cleft between the Walker A motif of one NBD and the ABC signature motif of the opposite NBD, triggering dimerization of two NBDs (Fig. 5e and Supplementary Fig. 9b, e). Both the observed conformational changes and the ATP-binding site are highly conserved in ABC transporters44,45. Hydrogen bonds and salt bridges are formed between ATP and the backbones or side chains of several key residues, including V738 and S741 of NBD1, as well as Y1251, G1281, K1282, S1283, S1284, and Q1324 of NBD2 (Fig. 5e). The conformational changes of the NBDs are transmitted to the TMBs via the linker helices located between the TMBs and NBDs (Fig. 5b–d). Consequently, the two TMBs pack closely on the intracellular side, sealing the substrate-binding cavity (Fig. 5f). In this state, the binding pocket is occluded on both sides of the membrane (Fig. 5f), representing an intermediate conformation of AtABCC2 following substrate release. Structural alignment of the monomeric apo-state and ATP-bound AtABCC2 structures revealed that while TMB2 aligns well in the two structures, the intracellular parts of TMB1 move toward TMB2. Large shifts are observed in TM6-TM8, TM11, TM15, and TM16 (Fig. 5g). Notably, TM11 undergoes a large conformational change, breaking from a long helix in the apo state into two helical segments. Two proline residues, P569 and P574, are located in the discontinuous region, possibly contributing to the helix breaking (Fig. 5h). On the cytosolic side of the TMBs, intense hydrogen bonds form between the two TMBs upon closure of the substrate-binding pocket, including interactions between S409 of TM8 and K1022 of TM14, S1093 of TM15 and N1018 of TM14, R589 of TM11, N401 of TM8 and D475 of TM9 (Supplementary Fig. 14a). On the vacuolar side, bulky hydrophobic residues seal the binding pocket, including L323 and L327 of TM6, F557 of TM11, and L1187 of TM17 (Supplementary Fig. 14b). Taken together, these two gates regulate the substrate entry from the cytosolic side and exit into the vacuolar side.

Methods
Protein expression and purification of AtABCC2
The DNA sequence of full-length WT AtABCC2, E1404Q mutant, and two truncated mutants ΔTMD0 (with amino acid residues 1–202 deleted) and ΔTMD0+L0 (with amino acid residues 1–265 deleted) were subcloned into a pCAG vector (Invitrogen) with an N-terminal Streptag and Flag tag. All AtABCC2 mutants were generated in the same way. A list of the primers used is provided in Supplementary Data 1. The HEK293F cells (Thermo Fisher Scientific, catalog number: A14635) were transfected with plasmids at a density of 2.5×106 cells mL−1. For 1 liter cell culture, 1.5 mg plasmids were premixed with 4 mg linear polyethylenimines (PEIs) (Polysciences) in 45 mL fresh medium for 15 min. Then the mixture was added into the cell, followed by a 15 min incubation. After 12 h, 10 mM sodium butyrate was added to the cells cultured at 30 °C. 48 h after transfection, the cells were collected by centrifugation at 2,100×g for 10 min. For protein purified in detergent digitonin and subjected to further biochemical and EM analysis, the cell pellet was resuspended in buffer containing 25 mM HEPES-HCl, pH 7.4, and 150 mM NaCl, supplemented with 1.5% (w/v) n-dodecyl-β-D-maltoside (DDM; Anatrace), 1 mM phenylmethylsulfonyl fluoride (PMSF), aprotinin (1.3 mg mL−1), and pepstatin (0.7 mg mL−1). The cell pellet was then incubated for 2 h at 4 °C by gentle agitation. After detergent extraction, the supernatant was collected by centrifugation at 200,000×g for 1 h and incubated with the anti-FLAG M2 affinity gel (Sigma-Aldrich) at 4 °C for 30 min. The resin was washed with 40 mL buffer containing 25 mM HEPES-HCl, pH 7.4, 150 mM NaCl, and 0.06% (w/v) digitonin (Apollo Scientific). Protein was eluted with the wash buffer plus 200 μg mL−1 FLAG peptides, which was then concentrated with a 100 kDa cut-off Centricon (Millipore). Finally, the sample was subjected to size-exclusion chromatography using the Superose-6 Increase column (GE Healthcare) in the buffer containing 25 mM HEPES-HCl, pH 7.4, 150 mM NaCl, and 0.03% (w/v) digitonin (Apollo Scientific). Peak fractions were collected and concentrated using a 100 kDa cut-off Centricon (Millipore) to ~16 mg mL−1 for further analysis.

For protein purified in detergent lauryl maltose neopentyl glycol (LMNG) plus cholesteryl hemisuccinate (CHS), the cell pellet resuspended buffer containing 25 mM HEPES-HCl, pH 7.4, and 150 mM NaCl, supplemented with 1% (w/v) LMNG plus 0.2% CHS (Anatrace), 1 mM phenylmethylsulfonyl fluoride (PMSF), aprotinin (1.3 mg mL−1), and pepstatin (0.7 mg mL−1). The wash buffer contains 25 mM HEPES-HCl, pH 7.4, and 150 mM NaCl, supplemented with 0.01% (w/v) LMNG and 0.002% CHS (Anatrace). The buffer of gel filtration contains 25 mM HEPES-HCl, pH 7.4, and 150 mM NaCl, supplemented with 0.005% (w/v) LMNG and 0.001% CHS (Anatrace). The other purification steps are the same as described above.

ATP hydrolysis measurements
The ATPase activity was measured using the ATPase Colorimetric Assay Kit (Innova Biosciences). 1 µg purified protein was added to the reaction buffer containing 25 mM HEPES-KOH (pH 7.4), 150 mM KCl, 10 mM MgCl , and 0.01% (w/v) DDM. Each substrate was diluted to serial concentrations, added to the mixture, and incubated for 20 min on ice. Next, ATP was added to the solution at a final concentration of 3 mM to start the reaction at 37 °C for 10 min, and the amount of inorganic phosphate released was measured quantitatively with the ATPase Colorimetric Assay Kit by determining the light absorbance at 650 nm wavelength with the BioTek Synergy H1 Multimode Reader. S-(2,4-dinitrophenyl)glutathione (DNP-GS), 17β-estradiol-β-D-glucuronide (E 17βG), used for the measurement of the concentration-dependent ATPase activity, were dissolved in DMSO and then serially diluted to the desired final concentrations in 1% (v/v) DMSO. β-D-glucopyranosyl abscisate (ABA-GE), GSH, GSSG, phytochelatin 2 (PC ) were dissolved in aqueous solution, and the final concentration is 60 μM. Statistical analysis was done using GraphPad Prism 10. Kinetics of the ATPase activities of AtABCC2 under serial concentrations of ligands were fitted with the Hill equation, with the Hill coefficient value fixed to 1.

Mass photometry
Mass photometry measurements were carried out on the TwoMP instrument (Refeyn Inc.). The glass cover slip (1.5H thickness, 24×50 mm, VWR) was cleaned consecutively with isopropanol and water, then dried using a stream of clean nitrogen. Resuspend the molecular weight standard in its matching buffer to calibrate the particle-image contrast caused by scattered light and convert it to molecular weight units. AtABCC2 , ΔTMD0, and AtABCC2 were diluted with the buffer used in size-exclusion chromatography to a final concentration of 1 μM. 1 μL of 1 μM protein solution was mixed with 15 μL buffer in the silicon gasket wells to 62.5 nM (the final concentration in the wells), and measurements were collected for 60 s. For each sample, the data was fitted with the Gaussian equation.

Sample preparation and cryo-EM data acquisition
The wild-type and E1404Q mutant AtABCC2 were concentrated to 16 mg mL−1 for cryo-EM sample preparation, respectively. After centrifugation at 11,000×g for 3 min, 4 μL of the protein sample was applied to the glow-discharged holey carbon grids (Quantifoil Au R1.2/1.3, 300 mesh). The grid was blotted using Vitrobot Mark IV (Thermo Fisher Scientific) operated at 8 °C and 100% humidity with a blotting time of 3 s. To prepare the complex sample, 10 mM ATP dissolved in purification buffer, or 800 μM DNP-GS dissolved in 2% DMSO, was incubated with apo-form AtABCC2 for 30 min at 4 °C before freezing grids. Micrographs were acquired on a Titan Krios (Thermo Fisher Scientific) electron microscope operating at 300 kV, equipped with the BioQuantum energy filter and a K3 direct electron detector (Gatan). Image stacks were recorded using EPU in the super-resolution mode with defocus values varied from −1.5 to −2.3 μm, an exposure time of 3 s, and dose-fractionated to 32 frames with a total dose of 50 e− Å−2.

Cryo-EM data processing
Motion correction and dose weighting were performed using the RELION 3.1 implementation of MotionCor255. Defocus values were estimated by CTFFIND456. For apo AtABCC2 , a total of 1,177,569 particles were automatically picked from 2123 micrographs and then subjected to 2D classification by using cryoSPARC (v3.2.0)57. In total, 471,775 particles from the best classes were put into ab initio reconstruction and non-uniform refinement, yielding a reconstruction monomeric map at an average resolution of 3.8 Å and a reconstruction dimeric map at an average resolution of 4.2 Å. For apo AtABCC2 , a total of 842,465 particles were automatically picked from 2205 micrographs and then subjected to 2D classification by using cryoSPARC (v.3.2.0). In total, 693,524 particles from the best classes were put into ab initio reconstruction and non-uniform refinement, yielding a reconstruction map at an average resolution of 3.5 Å. For the DNP-GS-bound AtABCC2 , a total of 1,270,918 particles were automatically picked from 2170 micrographs and then subjected to heterogeneous refinement and ab initio reconstruction. In total, 926,096 particles from the best classes were applied to ab initio reconstruction and non-uniform refinement. After multi-round refinement, yielding a final dimeric reconstruction map at an average resolution of 3.5 Å and a monomeric map at 4.1 Å. For the ATP-bound AtABCC2 , a total of 1,177,569 particles were automatically picked from 3027 micrographs and subjected to further 2D classification. In total, 1,116,364 particles from the best classes were applied to ab initio reconstruction and heterogeneous refinement, yielding a map with an overall resolution of 3.4 Å.

Model building and refinement
The final local refinement map was used for model building in Coot58,59. Using UCSF Chimera60,61, the initial rigid body model of AtABCC2 predicted by AlphaFold262 was docked into the construction map. Then, model building and refinement were performed manually by Coot. Structure refinements were carried out by PHENIX63 in real space. Overfitting of the model was monitored by refining the model in one of the two independent maps from the gold-standard refinement approach and testing the refined model against the other map. EM map or structure figures are prepared using PyMol or UCSF ChimeraX. The statistics pertaining to the 3D reconstruction and model refinement can be found in Supplementary Table 1.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Xuan and colleagues investigate the working mechanism of ABCC2 from Arabidopsis thaliana using structural and biochemical approaches. AtABCC2, located on the vacuolar membrane and belonging to the ATP-binding cassette (ABC) transporter superfamily, plays a crucial role in detoxification by exporting a variety of toxic substances. The authors determined several cryo-EM structures of AtABCC2 at different functional states, including the apo state (without substrate bound), the substrate DNP-GS bound state, and the ATP-bound state. Notably, in all three states, the authors identified both monomeric and dimeric ABCC2, although the ratio between monomer and dimer varies. With key residues identified through structural analysis, the authors further confirmed their critical roles via functional assays that combined mutagenesis and ATPase activity assays.\nOverall, this study provides valuable insights into the structural and functional aspects of AtABCC2 in mediating the transport of a wide range of substrates, enhancing our understanding of the detoxification processes in plants facilitated by the versatile ABCC2 transporter.\nHere are my comments:\n1. There are some duplicated descriptions in the introduction. For example, lines 68-72 introduce the “long” and “short” proteins in the ABCC subfamily.\n2. It is interesting to observe both monomeric and dimeric states of ABCC2 in all conditions. For the DNP-GS bound dimeric structure, did the authors observe one monomer occupied by the structure while the other was not? What is the physiological relevance of the oligomeric states of ABCC2? Will this facilitate substrate transport?\n3. The DNP-GS is stabilized by the amphipathic cavity. It would be beneficial if the authors could show a hydrophobic surface next to the electrostatic surface.\n4. The author should reorder the figures as well as extended data figures to ensure their sequence matches the order in which they are cited in the main text.\n5. Including a secondary structure topology in Figure 1 would help readers understand the domain organization of ABCC2 more easily.\n6. The unit of Vmax in the main text should be “nmol·min-1· mg-1” rather than “nmol-1min-1mg”.\n7. The term should be “cryo-EM density map” rather than “electron density map”, as this applies specifically to maps determined using single-particle analysis.\n8. In Extended Data Fig. 1, it would be better to place ABCC2-ARATH at the top since the structural segments are based on ABCC2 structure.\n9. In Extended Data Fig. 3, please indicate the peak fractions used for cryo-sample preparation.\n10. In Extended Data Fig. 3f, please maintain local resolution scale for two maps: high-resolution to low-resolution from left to right.\n11. In Extended Data Fig. 4b, please specify whether the densities are from monomeric or dimeric ABCC2 structures.\n12. In Extended Data Fig. 5e, it appears the rotation angle is incorrect. Similar inaccuracies are present in some main figures and other Extended Data Figs.\n13. In Extended Data Fig. 12, please double-check the rotation angles.\n14. In Extended Data Table 1, please verify the “Model Resolution” and “FSC Threshold” in “Model Refinement” section.", "reviewer_2": "This paper describes cryo-EM structures of ABCC2 from Arabidopsis thaliana (AtABCC2) in the absence and present of ATP or substrate. Notably, the structure of AtABCC2differs from other ABCC proteins in the location of TMD0. Additionally, AtABCC2 dimerizes, and this paper present the 3rd structure determined for ABCC dimers. Given the difference in the location of TMD0 in AtABCC2, the dimerization interface also differs from other ABCC proteins. The authors also present ATPase data in the absence and presence of substrate, and with deletion of TMD0 or TMD0 and the L0 linker. The deletion studies show that TMD0 is dispensable for ATPase activity, but the L0 is needed. While the study shows novel findings that would be of interest to the ABC transporter field, the following should be addressed before publication.\nMajor points\n1. The introduction comments on differences in the sequences of ABCC transporters across different species and shows a sequence alignment in Extended Data Fig. 1.\nFirst, the sequence alignment is missing part of the YCF1 protein sequence, and thus there are no residues for h1 for this protein. The authors should include the full sequence of Ycf1p.\nAdditionally, the figure legend indicates that the sequences were aligned with ClustalW, a program that offers the possibility of also using structural information in the alignment. Did the authors do this? If not, why?\nGiven the comments by the authors of the sequence differences between ABCC proteins in different species, a % sequence identity should be included. Additionally, the authors only include YCF1, bovine ABCC1, and human ABCC3 along with AtABCC2 in the alignment. Why not include other ABCC2 proteins, including human ABCC2 for which a high-resolution structure exists that could be compared to the AtABCC2 structure.\n2. ATPase measurements are shown in Fig. 1a for the WT type protein in absence of any substrates. However it isn’t clear whether these measurements are made on monomeric or dimeric protein, considering the associated Extended Data Fig. 6 shows evidence of monomeric and dimeric species. It is assumed that the ATPase data was acquired on a sample containing a mixture of species. If so, why were separate fractions not purified and assayed? If the monomer and dimer species have different ATPase activities, interpretation of one Km and Vmax value, is difficult. This is also important to address given that ATP-binding does not result in dissociation of the dimer.\nA similar situation occurs for ATPase assays with the truncation mutants ( TMD0 and  TMD0-L0). Additionally, do the truncations affect the level of dimerization? Considering the these elements are not involved in the dimerization, one would assume not, but this should be tested and/or explained, again because one set of ATPase measurements are presented for a monomer/dimer mixture.\nBecause removal of TMD0 does not affect activity, can the authors surmise what the role of TMD0 could be? Is there are role for TMD0 in mammalian ABCC2 discerned, for example?\n3. The size exclusion trace in Extended Data Fig. 6 needs some labels. Does the peak at 10 mL correspond an aggregate or the dimer? Or do the monomer and dimer elute in the broad peak at 15 mL. The fractions from the size exclusion column used for the accompanying gel should be indicate. Additionally, the gel should be destained further so that the reader gets a sense of the purity of the protein used for cryo-EM and for these assays.\n4. The authors present structures of WT ABCC2 in the monomeric and dimeric forms. However, the associated cryo-EM needs better presentation. First, only 2 classes for the monomer and dimer are shown. One would assume that there were more and at least some should be shown. Second, FSC curves and Euler angle distribution plots are shown only for the monomer. They should also be shown for the dimer.\n5. Additionally, the model-in-map fits in Extended Data Fig.4 (and in all other Extended Data Figs) need to be of higher resolution. It was hard for this reviewer to analyze the quality of the structures from these figures, particularly since only PDBs and not density files were provided. It is also unclear whether the model-in-map fits in Extended Data Fig.4 are for the monomer or dimer? I assume the monomer, but it is important to show also the dimer model-in-map fits, particularly as many if not all side chains are included in the ABCC dimer atomic model. Additionally, any figure in the main text where side chains are shown should have the associated density also shown. This includes Fig. 2f that show hydrophobic and polar contacts between protomers in the ABCC2 WT dimer.\n6. The location of TMD0 in ABCC2 is very interesting considering its location between TMD1 and TMD2, and not just at TMD1, as in other ABCC proteins. The authors compare the location of TMD0 in AtABCC2 to ABCC3, but they should also compare to human ABCC2, especially since the ABCC2 was determined in detergent micelles and not nanodiscs as in the case of ABBC3. The location of the ABCC2 TMD0 is somewhat reminiscent of the TMD0 in ABCB6, a homodimeric half-transporter where each protomer contains the canonical TMD-NBD, plus an N-terminal TMD0. Although TMD0 is at low resolution in the ABCB6 stucture, a comparison of the locations of ABCB6 TMD0 and the AtABCC2 TMD0 presented here could be made.\n7. The authors compare the structure of the dimeric AtABCC2 to that of YCF1, but they should also compare to dimeric MRP1 (or at least mention that such as structure exists, as it is similar to that of YCF1). In addition to discussing the different dimer interfaces, the authors should make other comparisons – e.g., buried surface areas, as it seems that the AtABCC2 buried surface area is larger; lipids at the interface, as these were seen both for YCF1 and MRP1, and in dimers of other membrane proteins.\n8. Going back to the sequence differences between ABCC proteins in different species introduced by the authors: can the authors rationalize the different TMD0 interactions in AtABCC2 vs human ABCC2, YCF1, MRP1, ABCC3 based on sequence, perhaps using evolution covariance? This may also yield insights into the different dimerization modes.\n9. The authors have also used a mutation of ABCC2 with the catalytic Glu in NBD2 substituted with a Gln. In contrast to the WT ABCC2, 75% of these particles were dimeric. However, isn’t possible that monomeric ABCC2 particles have worse contrast, considering that they are smaller, and thus not as easily picked? Are there independent biochemical measurements of monomer and dimer relative quantities? The size exclusion profiles for the WT and E/Q mutant ABCC2 purifications are very similar.\n10. The authors do not comment on the potential functions of the dimer in plant detoxification, just that it remains to be elucidated. They may wish to elaborate on possible functions, perhaps by looking at biochemical data showing oligomerization of other ABC proteins.\nMinor point\n1. Line 65 “is” should be “are”.\n2. The legend for Extended Data Fig. 1 should state that highly conserved residues are coloured red, in addition to the statement indicating the red shaded residues are invariant.\n3. Extended Data Figs. are not called out in order and should be. For example, Extended Data Fig. 3a is called out before Extended Data Fig. 2. Extended Data Fig. 6a is called out before Extended Data Figs. 3b-f, 4, and 5.\n4. Line 293: “extracellular” should be “vacuolar”."}, "author_response": "Response: We thank the reviewer for the positive feedback on our work, and for the constructive and helpful comments. The raised points are addressed below:\nHere are my comments:\n1. There are some duplicated descriptions in the introduction. For example, lines 68-72 introduce the “long” and “short” proteins in the ABCC subfamily.\nResponse: Thanks for pointing this out. We have removed such duplicated descriptions in the introduction section of the manuscript.\n2. It is interesting to observe both monomeric and dimeric states of ABCC2 in all conditions. For the DNP-GS bound dimeric structure, did the authors observe one monomer occupied by the structure while the other was not? What is the physiological relevance of the oligomeric states of ABCC2? Will this facilitate substrate transport?\nResponse: Thanks for the suggestion. For the DNP-GS bound dimeric structure, we have carried out 3D classification and reconstruction with the C1 symmetry. However, in the density maps, we did not observe that only one monomer was bound with DNP-GS while the other not.\nThe physiological relevance of the different oligomeric states of AtABCC2 is indeed an intriguing question. The existence of these different states in AtABCC2 were further validated by the mass photometry analysis results which we have added in the revised manuscript (Supplementary Fig. S7, and also see below). To examine the activity of the monomeric and dimeric forms, we tried to separate these different species in gel filtration. Since in the detergent digitonin, only a single peak could be observed in gel filtration for the WT AtABCC2, which make it hard to separate the monomer and dimer fractions, we tried other different conditions for the protein purification. After all the trials, the best condition we could get to separate the two species was to use detergent combination of LMNG and CHS during protein purification, which is also the detergent used to purify human ABCC1 as reported (Shinde et al., Immunity, 2025.). We have to admit that even under this condition, the two peaks were not largely separated. Thus, we collected the earlier fractions of peak-1 and the late fractions of peak2 to separate the two species as much as possible. Moreover, fractions of each peak were subjected to mass photometry analysis. The first peak predominantly (approximately 83%) had a molecular weight of 425 kDa corresponding to the dimeric form (Supplementary Fig. S7d and also see below), while the second peak predominantly (approximately 80%) had a molecular weight of 248 kDa corresponding to the monomeric form (Supplementary Fig. S7e and also see below). We then tested the ATPase activity of the two species. No significant differences were observed between the monomeric and dimeric species either with or without the substrate DNP-GS (Supplementary Fig. S7f, and also see below). Notably, the basal activity was relatively much higher for either the monomeric or dimeric AtABCC2 compared to that reported for Ycf1 or hABCC1 (Bickers et al., Nature Communications, 2024; Shinde et al., Immunity, 2025.). The monomeric to dimeric form transition of Ycf1p has been shown to be regulated by the protease cleavage, and the dimeric form has lower ATPase activity than the monomeric form (Bickers et al., Nature Communications, 2024.). For human ABCC1, the dimeric form also has lower ATPase activity in the absence of substrate, but its ATPase activity could be more significantly stimulated by the substrate than that of the monomeric form (Shinde et al., Immunity, 2025.). Since AtABCC2 adopts a distinct dimer organization compared to Ycf1p and human ABCC1, and ATP binding does not lead to disassembly of the dimer as we have observed in the structures, it indicates that AtABCC2 may have different regulation mechanism for the assembly of dimeric and monomeric states or in vivo activities. The physiological meaning of the dimeric and monomeric species of AtABCC2 remains to be further investigated. In the revised manuscript, we have added the above results and a discussion about the possible regulation of the monomeric and dimeric forms of AtABCC2.\n3. The DNP-GS is stabilized by the amphipathic cavity. It would be beneficial if the authors could show a hydrophobic surface next to the electrostatic surface.\nResponse: Thanks for the suggestion. In the revised manuscript, we have added a panel showing the hydrophobic surface of the DNP-GS binding cavity to Figure 4 (Fig. 4d of the revised manuscript, and also see below).\n4. The author should reorder the figures as well as extended data figures to ensure their sequence matches the order in which they are cited in the main text.\nResponse: Thanks for the suggestion. We have reordered the figures to match the order citated in the main text.\n5. Including a secondary structure topology in Figure 1 would help readers understand the domain organization of ABCC2 more easily.\nResponse: Thanks for the suggestion. We have added a secondary structure topology in Figure 2 following the 3D structure of AtABCC2 (Fig. 2c of the revised manuscript, and also see below).\n6. The unit of Vmax in the main text should be “nmol·min-1· mg-1” rather than “nmol-1min-1mg”.\nResponse: Thanks for pointing this out. We have corrected this typo in the revised manuscript.\n7. The term should be “cryo-EM density map” rather than “electron density map”, as this applies specifically to maps determined using single-particle analysis.\nResponse: Thanks for pointing this out. We have corrected this in the revised manuscript.\n8. In Extended Data Fig. 1, it would be better to place ABCC2-ARATH at the top since the structural segments are based on ABCC2 structure.\nResponse: Thanks for the suggestion. We have reordered the sequences to place AtABCC2 at the top in the revised Supplementary Figs. S1 and S2.\n9. In Extended Data Fig. 3, please indicate the peak fractions used for cryo-sample preparation.\nResponse: Thanks for the suggestion. We have indicated the peak fractions used for cryo-sample preparation in the revised Supplementary Fig. S3a, b.\n10. In Extended Data Fig. 3f, please maintain local resolution scale for two maps: high-resolution to low-resolution from left to right.\nResponse: Thanks for the suggestion. We have revised the panes as suggested.\n11. In Extended Data Fig. 4b, please specify whether the densities are from monomeric or dimeric ABCC2 structures.\nResponse: Thanks for the suggestion. We have clarified this point in the revised figures including Supplementary Figs. S4 and S5.\n12. In Extended Data Fig. 5e, it appears the rotation angle is incorrect. Similar inaccuracies are present in some main figures and other Extended Data Figs.\nResponse: Thanks for pointing this out. We have corrected this error in the revised Supplementary Figures and main figures.\n13. In Extended Data Fig. 12, please double-check the rotation angles.\nResponse: Thanks for pointing this out. We have corrected this point in the revised figure.\n14. In Extended Data Table 1, please verify the “Model Resolution” and “FSC Threshold” in “Model Refinement” section.\nResponse: Thanks for pointing this out. We have corrected the model resolution and FSC threshold in the Supplementary Table S1.\nReviewer #2:\nThis paper describes cryo-EM structures of ABCC2 from Arabidopsis thaliana (AtABCC2) in the absence and present of ATP or substrate. Notably, the structure of AtABCC2differs from other ABCC proteins in the location of TMD0. Additionally, AtABCC2 dimerizes, and this paper present the 3rd structure determined for ABCC dimers. Given the difference in the location of TMD0 in AtABCC2, the dimerization interface also differs from other ABCC proteins. The authors also present ATPase data in the absence and presence of substrate, and with deletion of TMD0 or TMD0 and the L0 linker. The deletion studies show that TMD0 is dispensable for ATPase activity, but the L0 is needed. While the study shows novel findings that would be of interest to the ABC transporter field, the following should be addressed before publication.\nResponse: We thank the reviewer for the positive feedback on our work and for the constructive and helpful comments. The raised points are addressed below:\nMajor points：\n1. The introduction comments on differences in the sequences of ABCC transporters across different species and shows a sequence alignment in Extended Data Fig. 1.\nFirst, the sequence alignment is missing part of the YCF1 protein sequence, and thus there are no residues for h1 for this protein. The authors should include the full sequence of Ycf1p.\nResponse: Thanks for the suggestion. We have included the full sequences of Ycf1, AtABCC2, human ABCC1-3, and bovine ABCC1 in the sequence alignment. Due to the long length of the sequences, we have divided the results into two separate figures, one containing the N-terminal half and the other containing the C-terminal half (Supplementary Figs. S1 and S2a of the revised manuscript).\nAdditionally, the figure legend indicates that the sequences were aligned with ClustalW, a program that offers the possibility of also using structural information in the alignment. Did the authors do this? If not, why?\nThe sequence alignment was performed using the ClustalW program. For this program, to our knowledge, it could not use the structural information for sequence alignment. However, as we learned that, in the software ChimeraX, the pairwise sequence alignments can be shown after structure alignments. But still only two sequences could be shown. Thus, we performed the structure alignment of Ycf1 and AtABCC2 as an example, and analyzed the resulting sequence alignment (see below). The structure-based alignment results are quite similar to the sequence alignment results generated using ClustalW. Considering that not all the sequences were modelled in the structures and some parts are missing in the results generated in ChimeraX, we presented the sequence alignment results by ClustalW as shown in Supplementary Figs. S1 and S2a of the revised manuscript.\nGiven the comments by the authors of the sequence differences between ABCC proteins in different species, a % sequence identity should be included. Additionally, the authors only include YCF1, bovine ABCC1, and human ABCC3 along with AtABCC2 in the alignment. Why not include other ABCC2 proteins, including human ABCC2 for which a high-resolution structure exists that could be compared to the AtABCC2 structure.\nWe are sorry for missing the human ABCC2 in the sequence alignment. In the revised manuscript, we have included human ABCC2 now. As suggested by the reviewer, we also added a panel of the percentage of sequence identity in the revised manuscript (Supplementary Fig. S2b of the revised manuscript).\n2. ATPase measurements are shown in Fig. 1a for the WT type protein in absence of any substrates. However, it isn’t clear whether these measurements are made on monomeric or dimeric protein, considering the associated Extended Data Fig. 6 shows evidence of monomeric and dimeric species. It is assumed that the ATPase data was acquired on a sample containing a mixture of species. If so, why were separate fractions not purified and assayed? If the monomer and dimer species have different ATPase activities, interpretation of one Km and Vmax value, is difficult. This is also important to address given that ATP-binding does not result in dissociation of the dimer.\nResponse: Thanks for the suggestion. As shown by the cryo-EM analysis results, both monomeric and dimeric forms of AtABCC2 exist in the purified protein in detergent digitonin, with a ratio of monomer to dimer of approximately 2:1. To further confirm this, we also used the mass photometry method to analyze the protein sample. As shown by the results, in the wild-type AtABCC2, both species with molecular weights corresponding to the monomeric and dimeric forms were observed, with a ratio of approximately 2:1 of monomer to dimer, consistent with the cryo-EM analysis (Supplementary Fig. S7 and also see below).\nTo examine the activity of the monomeric and dimeric forms, we tried to separate these different species in gel filtration. Since in the detergent digitonin, only a single peak could be observed in gel filtration for the WT AtABCC2, which make it hard to separate the monomer and dimer fractions, we tried other different conditions for the protein purification. After all the trials, the best condition we could get to separate the two species was to use detergent combination of LMNG and CHS during protein purification, which is also the detergent used to purify human ABCC1 as reported (Shinde et al., Immunity, 2025.). We have to admit that, even under this condition, the two peaks were not largely separated. Thus, we collected the earlier fractions of peak-1 and the late fractions of peak2 to separate the two species as much as possible. Moreover, fractions of each peak were subjected to mass photometry analysis. The first peak predominantly (approximately 83%) had a molecular weight of 425 kDa corresponding to the dimeric form (Supplementary Fig. S7d and also see below), while the second peak predominantly (approximately 80%) had a molecular weight of 248 kDa corresponding to the monomeric form (Supplementary Fig. S7e and also see below). We then tested the ATPase activity of the two species. No significant differences were observed between the monomeric and dimeric species either with or without the substrate DNP-GS (Supplementary Fig. S7f and also see below).\nNotably, the basal activity was relatively much higher for either the monomeric or dimeric AtABCC2 compared to that reported for Ycf1 or hABCC1 (Bickers et al., Nature Communications, 2024; Shinde et al., Immunity, 2025.). The monomeric to dimeric form transition of Ycf1p has been shown to be regulated by the protease cleavage, and the dimeric form has lower ATPase activity than the monomeric form (Bickers et al., Nature Communications, 2024.). For human ABCC1, the dimeric form also has lower ATPase activity in the absence of substrate, but its ATPase activity could be more significantly stimulated by the substrate than that of the monomeric form (Shinde et al., Immunity, 2025.). Since AtABCC2 adopts a distinct dimer organization compared to Ycf1p and human ABCC1, and ATP binding does not lead to disassembly of the dimer as we have observed in the structures, it indicates that AtABCC2 may have different regulation mechanism for the assembly of dimeric and monomeric states or in vivo activities. The physiological meaning of the dimeric and monomeric species of AtABCC2 remains to be further investigated. In the revised manuscript, we have added the above results and a discussion about the possible regulation of the monomeric and dimeric forms of AtABCC2.\nA similar situation occurs for ATPase assays with the truncation mutants (ΔTMD0 and ΔTMD0-L0). Additionally, do the truncations affect the level of dimerization? Considering these elements are not involved in the dimerization, one would assume not, but this should be tested and/or explained, again because one set of ATPase measurements are presented for a monomer/dimer mixture.\nThanks for the suggestion. We also checked the oligomerization state of the ΔTMD0 mutant using mass photometry. As shown by the results (Supplementary Fig. S7a of the revised manuscript, and also see above), the ΔTMD0 mutant also exists in both dimeric and monomeric forms, quite similar to the wild-type AtABCC2, indicating that the truncation does not affect its dimerization. This is consistent with the structural observation that TMD0 is not involved in the formation of the dimer interface of AtABCC2. Since the monomeric and dimeric forms of the wild-type AtABCC2 showed no significant differences in the ATPase assay, we did not further characterize the different forms of the truncation mutants.\nBecause removal of TMD0 does not affect activity, can the authors surmise what the role of TMD0 could be? Is there are role for TMD0 in mammalian ABCC2 discerned, for example?\nThanks for the suggestion. Previous studies have shown that the N-terminal TMD0 domain of the ABCC transporters may play a crucial role in the proper localization of the protein, their interactions with other regulatory factors, and the regulation of the transport activity. For example, in human ABCC2, it is reported that TMD0 is essential to its correct apical membrane localization. In human ABCC8 (Sulfonylurea Receptor 1, SUR1) or ABCC9 (SUR2), the TMD0 domain mediates the interaction with the inward rectifier potassium channel subunit (Kir6.1 or Kir6.2), and together form the ATP-sensitive potassium channel (KATP). Whether TMD0 of AtABCC2 has a similar role in plants remain further in vivo investigations. We have added a discussion about this point in the revised manuscript.\n3. The size exclusion trace in Extended Data Fig. 6 needs some labels. Does the peak at 10 mL correspond an aggregate or the dimer? Or do the monomer and dimer elute in the broad peak at 15 mL. The fractions from the size exclusion column used for the accompanying gel should be indicate. Additionally, the gel should be destained further so that the reader gets a sense of the purity of the protein used for cryo-EM and for these assays.\nResponse: Thanks for the suggestion. We have further clarified the gel filtration results by adding more labels as suggested (Supplementary Fig. S3a, b of the revised manuscript, and also see below). The peak at 10 mL corresponds to an aggregate since no clear protein bands were observed in the SDS-PAGE, and the monomeric and dimeric forms elute in the broad peak at 15 mL which is difficult to separate under this condition. We have indicated the fractions from the size exclusion column used for the SDS-PAGE analysis and cryo-EM sample preparation in the revised figure. We also presented a much clearer SDS-PAGE result in the revised figure as suggested by the reviewer.\n4. The authors present structures of WT ABCC2 in the monomeric and dimeric forms. However, the associated cryo-EM needs better presentation. First, only 2 classes for the monomer and dimer are shown. One would assume that there were more and at least some should be shown. Second, FSC curves and Euler angle distribution plots are shown only for the monomer. They should also be shown for the dimer.\nResponse: Thanks for the suggestion. In the revised manuscript, we have shown more representative 2D class averages for the monomeric and dimeric forms. We also added the FSC curves and Euler angle distribution plots for both the monomer and the dimer (Supplementary Fig. 4 of the revised manuscript, and also see below).\n5. Additionally, the model-in-map fits in Extended Data Fig.4 (and in all other Extended Data Figs) need to be of higher resolution. It was hard for this reviewer to analyze the quality of the structures from these figures, particularly since only PDBs and not density files were provided. It is also unclear whether the model-in-map fits in Extended Data Fig.4 are for the monomer or dimer? I assume the monomer, but it is important to show also the dimer model-in-map fits, particularly as many if not all side chains are included in the ABCC dimer atomic model. Additionally, any figure in the main text where side chains are shown should have the associated density also shown. This includes Fig. 2f that show hydrophobic and polar contacts between protomers in the ABCC2 WT dimer.\nResponse: Thanks for the suggestion. We are sorry for the low-resolution images presented in the figures. In the revised manuscript, we have increased the resolution of the images. As shown in Supplementary Fig. 5b-c now (also see below), the sides chains fit well to the EM densities of the wild-type AtABCC2. Also, both model-in-maps of the monomer and dimer are shown. Specially, although the overall resolution of the EM map of the dimeric wild-type AtABCC2 was low, the densities for most side chains of TM1-TM12 could be clearly viewed and fit well.\nAlso as suggested by the reviewer, we have shown the associated EM density for the side chains presented in the main text including Fig. 2g and Fig. 3c. The EM densities for the side chains of the residues involved in substrate binding presented in Fig. 4d was shown in Supplementary Fig. 10f.\n6. The location of TMD0 in ABCC2 is very interesting considering its location between TMD1 and TMD2, and not just at TMD1, as in other ABCC proteins. The authors compare the location of TMD0 in AtABCC2 to ABCC3, but they should also compare to human ABCC2, especially since the ABCC2 was determined in detergent micelles and not nanodiscs as in the case of ABBC3. The location of the ABCC2 TMD0 is somewhat reminiscent of the TMD0 in ABCB6, a homodimeric half-transporter where each protomer contains the canonical TMD-NBD, plus an N-terminal TMD0. Although TMD0 is at low resolution in the ABCB6 structure, a comparison of the locations of ABCB6 TMD0 and the AtABCC2 TMD0 presented here could be made.\nResponse: Thanks for the suggestion. As suggested, we have also compared our structure with the human ABCC2 and ABCB6. As shown by the alignment, although the L0 linker of AtABCC2 aligned well with the ABCC homologues including hABCC2 and hABCC3, TMD0 exhibited a significant lateral shift (Fig. 2f of the revised manuscript, and also see below). In hABCC2 and hABCC3, TMD0 is positioned laterally relative to TMD1, whereas in AtABCC2, TMD0 is located between the two TMDs. It suggests that the extra N-terminal TMD0 domain displays structural variations among ABCC homologues. The TMD0 domains in hABCB6 and AtABCC2 also show different localizations relative to the core TMDs.\n7. The authors compare the structure of the dimeric AtABCC2 to that of YCF1, but they should also compare to dimeric MRP1 (or at least mention that such as structure exists, as it is similar to that of YCF1). In addition to discussing the different dimer interfaces, the authors should make other comparisons – e.g., buried surface areas, as it seems that the AtABCC2 buried surface area is larger; lipids at the interface, as these were seen both for YCF1 and MRP1, and in dimers of other membrane proteins.\nResponse: Thanks for the suggestion. In the revised manuscript, we have included the structure comparisons of AtABCC2 to both Ycf1 and human ABCC1 (or MRP1) (Supplementary Fig. S9b-e of the revised manuscript, and also see below). Ycf1 and hABCC1 adopt similar dimer architectures, with the dimer interface formed by both interactions between TMD0 and the core TMD and interactions mediated by lipid molecules. However, for AtABCC2, the dimer interface in the transmembrane region was formed by the core TMDs. Lipid molecules were also observed in the dimer interface of AtABCC2.\nWe also compared the buried surface areas in the three transporters. As shown by the results, Ycf1 has the largest buried surface area of 1599 Å2, while AtABCC2 and hABCC1 have similar buried surface areas. This is consistent with the structure observations that Ycf1 has a much compact dimeric structure compared to AtABCC2 and hABCC1. We have added these comparisons in the revised manuscript.\n8. Going back to the sequence differences between ABCC proteins in different species introduced by the authors: can the authors rationalize the different TMD0 interactions in AtABCC2 vs human ABCC2, YCF1, MRP1, ABCC3 based on sequence, perhaps using evolution covariance? This may also yield insights into the different dimerization modes.\nResponse: Thanks for the suggestion. As the sequence alignment results showed, the TMD0 sequence varies and has low conservation among AtABCC2, human ABCC1-ABCC3 and YCF1. To investigate possible evolution covariance, we used CoeViz program (Baker et al., BMC Bioinformatics, 2016.), a tool for coevolution analysis of protein residues, to analyze the AtABCC2 sequence and looked for possible pairwise coevolution of amino acid residues (see below). However, of the interacting residue pairs between TMD0 and the core TMD shown in Fig. 2g, no high covariance was observed. High covariance values were detected between A25-L1546, F26-L1402/L1448, V38-S1526, I41-D1444, and Y149-D1551. However, all these residues coupled to those in TMD0 are located in the NBD2 or the C-terminal region of AtABCC2. We also tried using EVcouplings to do the coevolution analysis. But still no high covariance was identified for these residues of TMD0. We are sorry for not being experts in such bioinformatic analysis to get more clues about the differences observed in the TMD0 or dimer architecture. Hope more information can be drawn from our AtABCC2 structures and the available ABCC homologues with more in-depth characterizations.\n9. The authors have also used a mutation of ABCC2 with the catalytic Glu in NBD2 substituted with a Gln. In contrast to the WT ABCC2, 75% of these particles were dimeric. However, isn’t possible that monomeric ABCC2 particles have worse contrast, considering that they are smaller, and thus not as easily picked? Are there independent biochemical measurements of monomer and dimer relative quantities? The size exclusion profiles for the WT and E/Q mutant ABCC2 purifications are very similar.\nResponse: Thanks for the suggestion. We went back to the cryo-EM datasets of the AtABCC2 EQ mutant, and carried out further 2D and 3D classifications of the particles. Indeed, there are small portions of particles corresponding to the monomeric AtABCC2 protein in both the apo state, the DNP-GS-bound state, and the ATP-bound state (Supplementary Figs. S8f, S10e, and S12e). However, due to the relatively small data size and low contrast, we could not get high resolutions structures of the EQ mutant in the apo state or the ATP-bound state. For the DNP-GS-bound state, we were able to obtain an EM map at an overall resolution of 4.1 Å. Docking of the monomeric map and the dimeric map showed that the maps merged well, suggesting almost identical conformation (inset panel of Supplementary Figs. S10e). Thus, we focus on the analysis of the dimeric structure. We have revised the data processing figures in the manuscript accordingly.\nAs we also carried out mass photometry analysis of the AtABCC2 EQ mutant sample, both monomeric and dimeric forms exist in the sample (Supplementary Fig. S7a and also see below). Unlike the wild-type protein, the dimeric form is the dominant state for the EQ mutant, consistent with the cryo-EM observations.\n10. The authors do not comment on the potential functions of the dimer in plant detoxification, just that it remains to be elucidated. They may wish to elaborate on possible functions, perhaps by looking at biochemical data showing oligomerization of other ABC proteins.\nResponse: Thanks for the suggestion. The dimeric structures and functions have been characterized for the yeast Ycf1 and human ABCC1. For Ycf1, the phosphorylation level of its regulatory region and ATPase activity are both down-regulated in the dimeric form than the monomeric form, suggesting that dimerization plays a role in its transport activity regulation (Bickers et al., Nature Communications, 2024). For human ABCC1, while the monomer has higher ATPase activity in the absence of substrate, the dimeric form’s ATPase activity can be significantly stimulated by the substrate cGAMP, indicating that the dimer is the active cGAMP transporter (Shinde et al., Immunity, 2025.). In the revised manuscript, we elaborate the discussion of possible functions of the dimeric form of AtABCC2 in plant, stating that possible regulation of its ATPase and transport activity, interactions with other regulators, and possible regulation by lipids. We have added this discussion in the revised manuscript.\nMinor point：\n1. Line 65 “is” should be “are”.\nResponse: Thanks for pointing this out. We have corrected this typo in the revised manuscript.\n2. The legend for Extended Data Fig. 1 should state that highly conserved residues are coloured red, in addition to the statement indicating the red shaded residues are invariant.\nResponse: Thanks for the suggestion. We have added the description in the figure legend.\n3. Extended Data Figs. are not called out in order and should be. For example, Extended Data Fig. 3a is called out before Extended Data Fig. 2. Extended Data Fig. 6a is called out before Extended Data Figs. 3b-f, 4, and 5.\nResponse: Thanks for the suggestion. We have reordered the figures to make sure they follow their order of calling out in the main text.\n4. Line 293: “extracellular” should be “vacuolar”.\nResponse: Thanks for pointing this out. We have corrected this in the revised manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In the revised manuscript, the authors have addressed all of my concerns raised in the previous review. Only a few minor points for consideration:\n1. Line 35: “… , can minimizing…”, should be “minimize”.\n2. Line 36-39: “Typically, … the cells” should be revised to avoid confusion and improve clarity for readers.\n3. Line 130. NBD has already been defined previously; there is no need to use the full name here.\n4. The authors may consider using either “protomer”, “subunit”, or “monomer” consistently throughout the manuscript to describe the structural features of dimeric AtABCC2.\n5. Line 498, 504, 506 and so on: please add a space between the number and the degree symbol.\n6. Line 556 and 557: “16 mg ml-1” and “4 μl” should be “mL-1” and “μL”, respectively.", "reviewer_2": "The revised manuscript contains additional experiments (mass photometry) studies that allowed the authors to address some of the original reviewers' comments, namely presenting additional data that shows the presence of two oligomeric states of AtABCC2 and measuring the ATPase activities of the monomer and dimer forms.\nHowever, there are some comments regarding presentation of the dimer structures that have not been addressed, and which need to be addressed before publication.\n1. I appreciate that the authors included the density for sidechains shown, as requested. However, they did this only for the monomeric structures (Figs. XXX), but not for the dimer structures (Fig. S6d and Fig. 3c show sidechains across protomers but no density.) Showing the cryo-EM density is critical to allow the reader to evaluate the structure.\n2. Speaking of evaluating the structures, there are 6 structures presented in this paper. The authors are commended for determining these various structures. However, structural reports are only provided for some. For example, Supplementary Table 1 has statistics for only 4 structures. The statistics for the Apo AtABCC2WT dimer and AtABCC2EQ+DNP-GS monomer structures are missing.\n3. Additionally, only 4 validation reports from the PDB are provided. Because there are no titles in the reports, I can not tell which report goes with which structure. Further, of the 4 reports provided, only 1 lists the resolution (of 3.4 Angstroms) which I assume is the AtABCC2EQ+ATP dimer structure given the resolutions reported in the Supplementary Figures. All of the validation reports (with resolutions) need to be provided in a manner that indicates which report goes with which structure.\nMinor points the authors should consider:\n1. The statement on lines 26-27 stating \"Conformational changes are observed upon binding of either substrate or ATP.\" make it seem like ATP binding causes drastic changes in the dimer, given the previous sentence. The authors should qualify what they mean here - i.e., movement of the TMDs and NBDs in the ABC core closer together to form the occluded state.\n2. On lines 72-74, the authors state \"However, the protein sequences vary significantly across species, particularly in the transmembrane region (Supplementary Figs. S1 and S2a, b).\" However, this is not reflected in the associated data. In fact, the sequence alignment shows sequence variability in TMD0 but that TMD1 and TMD2 sequences are relatively conserved.\n3. On lines 126 and 127, the authors describe the better resolution of the TMDs vs NBDs of their AtABCC2 wt structures. However, the vacuolar side of the TMDs are of lower resolution than the rest. Can the authors comment on the cause - loops, flexibility?\n4. The sentence on lines 132-133 needs to be altered. Currently it reads as follows: \"TMD1 is formed by TM1-TM3, TM6, TM10, and TM11, whereas TMD2 consists of TM4, TM5, TM7-TM9, and TM12 (Fig. 2b-d).\"\nHowever, In ABCC transporters with a TMD0, TMD1 is formed by TM6-11 TMD2 is formed by TM12-17. The TMD1/TMD2 domain swapping means that TM1-TM3, TM6, TM10, TM11 form one membrane structural unit. TM4, TM5, TM7-TM9, and TM12 form another structural unit.\nChanging this sentence is needed because in other parts of the paper, TMD1 is referred to as being formed my TM6-11 (e.g., lines 137-139 where TMD0 and L0 linker are described as preceeding TMD1 and Fig. 2c is called out showing the linear schematic of AtABCC2).\n5. The observation that AtABCC2 lacking substrate adopts an inward-facing conformation is typical of what is observed in other ABC transporters that have similar structures to AtABCC2. The authors should state this fact and provide appropriate references (e.g., review paper on ABC transporter structures).\n6. The callout to Fig, 3e on line 148 should be Fig. 2f, I think.\nAlso, while it is clear that the position of TMD0 changes in AtABCC2 compared to other ABCC proteins, this figure is hard to look at. One thing that might help is if the authors structurally aligned the TMD1/TMD2 of each transporter, which would simplify the figure as the TMD1/TMD2 helices would be superimposed.\nAdditionally, it might good include ABCB6 in a separate figure.\n7. On lines 153-155, the authors state that TMD0 packs against the ABC core \"primarily through hydrophobic packing and hydrogen bonding (Fig. 2g).\" However, the figure shows only hydrophobic amino acids at the interface. Where are the H-bonds?\n8. Do the authors know whether the deltaTMD0-L0 construct is still folded properly either via SEC or mass photomtry?\n9. The authors are commended for attempts to separate monomers and dimers with different detergents. It seems for the wildtype protein that the different detergents change the proportion of monomer/dimer. Can the authors comment on that?\n10. TMD2 in line 214 should be TMD1, as TM7 and TM8 are part of TMD1. This goes back to minor point #4 above.\n11. In the dimeric ABCC2EQ structure, the orientation of the NBDs changes. Can the authors comment on why the catalytic mutation changes the orientation of the NBDs, either through their structures or comparison with other work?\n12. Lines 317-319 describe where ATP binds at the NBDs in AtABCC2. The AtABCC2 ATP binding site is similar to that in other ABC transporters. The authors should state this and provide references.\n13. The authors also state (319-321) that a mixture of salt bridges and H-bonds form between ATP and the NBDs, listing V738 as one of the NBD1 residues that binds ATP. A Val can not form H-bonds, unless the backbone is involved. This should be clarified.\n14. The protein interactions shown in Fig. S13 should have the associated density plotted as well.\n15. The transport cycle proposed for AtABCC2 in the discussion is similar to that proposed for other ABC proteins. Any similarities should be stated and referenced, and any differences could be highlighted."}, "author_response": "Response: We thank the reviewer for the positive feedback on our work, and for the constructive and helpful comments. The raised minor points are addressed below:\n1. Line 35: “… , can minimizing…”, should be “minimize”.\nResponse: Thanks for pointing this out. We have corrected the error in the revised manuscript.\n2. Line 36-39: “Typically, … the cells” should be revised to avoid confusion and improve clarity for readers.\nResponse: Thanks for pointing this out. We have revised the sentence in the revised manuscript (lines 35-38).\n3. Line 130. NBD has already been defined previously; there is no need to use the full name here.\nResponse: Thanks for the suggestion. We have corrected this in the manuscript.\n4. The authors may consider using either “protomer”, “subunit”, or “monomer” consistently throughout the manuscript to describe the structural features of dimeric AtABCC2.\nResponse: Thanks for pointing this out. We have unified the nomenclature to “protomer” in the revised manuscript.\n5. Line 498, 504, 506 and so on: please add a space between the number and the degree symbol.\nResponse: Thanks for pointing this out. We have revised the expression in the manuscript.\n6. Line 556 and 557: “16 mg ml-1” and “4 μl” should be “mL-1” and “μL”, respectively.\nResponse: Thanks for pointing this out. We have corrected these expressions in the revised manuscript.\nReviewer #2:\nWe thank the reviewer for the positive feedback on our work and for the constructive and helpful comments. The raised points are addressed below:\n1. I appreciate that the authors included the density for sidechains shown, as requested. However, they did this only for the monomeric structures (Figs. XXX), but not for the dimer structures (Fig. S6d and Fig. 3c show sidechains across protomers but no density.) Showing the cryo-EM density is critical to allow the reader to evaluate the structure.\nResponse: Thanks for the suggestion. We have included the map density for the dimer structures presented in Fig. 3c and Fig. S6d in the revised manuscript.\n2. Speaking of evaluating the structures, there are 6 structures presented in this paper. The authors are commended for determining these various structures. However, structural reports are only provided for some. For example, Supplementary Table 1 has statistics for only 4 structures. The statistics for the Apo AtABCC2WT dimer and AtABCC2EQ+DNP-GS monomer structures are missing.\nResponse: Thanks for the suggestion. We have included the statistics for the Apo AtABCC2 dimer and the AtABCC2 +DNP-GS monomer structures in Supplementary WT EQ Table 1 of the revised manuscript.\n3. Additionally, only 4 validation reports from the PDB are provided. Because there are no titles in the reports, I can not tell which report goes with which structure. Further, of the 4 reports provided, only 1 lists the resolution (of 3.4 Angstroms) which I assume is the AtABCC2EQ+ATP dimer structure given the resolutions reported in the Supplementary Figures. All of the validation reports (with resolutions) need to be provided in a manner that indicates which report goes with which structure.\nResponse: Thanks for the suggestion. We have submitted all the 6 structures presented in the paper to PDB. The validation reports are now provided with the title included in the first page of the PDF files.\nMinor points the authors should consider:\n1. The statement on lines 26-27 stating \"Conformational changes are observed upon binding of either substrate or ATP.\" make it seem like ATP binding causes drastic changes in the dimer, given the previous sentence. The authors should qualify what they mean here - i.e., movement of the TMDs and NBDs in the ABC core closer together to form the occluded state.\nResponse: Thanks for the suggestion. We have revised this sentence according to the suggestion (lines 25-27).\n2. On lines 72-74, the authors state \"However, the protein sequences vary significantly across species, particularly in the transmembrane region (Supplementary Figs. S1 and S2a, b).\" However, this is not reflected in the associated data. In fact, the sequence alignment shows sequence variability in TMD0 but that TMD1 and TMD2 sequences are relatively conserved.\nResponse: Thanks for pointing this out. We have revised this sentence in the manuscript (lines 71-72).\n3. On lines 126 and 127, the authors describe the better resolution of the TMDs vs NBDs of their AtABCC2 structures. However, the vacuolar side of the TMDs are of lower WT resolution than the rest. Can the authors comment on the cause - loops, flexibility?\nResponse: Thanks for pointing this out. The reason for the lower resolution of the TMDs on the vacuolar side may be due to the intrinsic flexibility of this region, particularly considering possible relative movements of TMD0 and the core TMDs. We have added this notion in the revised manuscript (lines 126-128).\n4. The sentence on lines 132-133 needs to be altered. Currently it reads as follows: \"TMD1 is formed by TM1-TM3, TM6, TM10, and TM11, whereas TMD2 consists of TM4, TM5, TM7-TM9, and TM12 (Fig. 2b-d).\" However, In ABCC transporters with a TMD0, TMD1 is formed by TM6-11 TMD2 is formed by TM12-17. The TMD1/TMD2 domain swapping means that TM1-TM3, TM6, TM10, TM11 form one membrane structural unit. TM4, TM5, TM7-TM9, and TM12 form another structural unit. Changing this sentence is needed because in other parts of the paper, TMD1 is referred to as being formed by TM6-11 (e.g., lines 137-139 where TMD0 and L0 linker are described as preceding TMD1 and Fig. 2c is called out showing the linear schematic of AtABCC2).\nResponse: Thanks for pointing this out. We have revised this sentence in the manuscript (lines 131-135). We have also renumbered the transmembrane helices, TM1-TM5 for TMD0, TM6-TM11 for TMD1, and TM12-TM17 for TMD2. All figures are revised accordingly in the manuscript.\n5. The observation that AtABCC2 lacking substrate adopts an inward-facing conformation is typical of what is observed in other ABC transporters that have similar structures to AtABCC2. The authors should state this fact and provide appropriate references (e.g., review paper on ABC transporter structures).\nResponse: Thanks for pointing this out. We have added this point and the related references in the revised manuscript (lines 137-139).\n6. The callout to Fig, 3e on line 148 should be Fig. 2f, I think. Also, while it is clear that the position of TMD0 changes in AtABCC2 compared to other ABCC proteins, this figure is hard to look at. One thing that might help is if the authors structurally aligned the TMD1/TMD2 of each transporter, which would simplify the figure as the TMD1/TMD2 helices would be superimposed. Additionally, it might good include ABCB6 in a separate figure.\nResponse: Thanks for pointing this out. We have corrected this in the revised manuscript. We have also revised Fig. 2f according to the reviewer’s suggestion to align TMD1/TMD2 first. The alignment with hABCB6 is now shown in a separate figure as in Supplementary Fig. 6a.\n7. On lines 153-155, the authors state that TMD0 packs against the ABC core \"primarily through hydrophobic packing and hydrogen bonding (Fig. 2g).\" However, the figure shows only hydrophobic amino acids at the interface. Where are the H-bonds?\nResponse: Thanks for pointing this out. In Fig. 2g, we show the hydrogen bonds with red dashed lines, like between backbone of A25 and R1168, between backbones of A25 and M1164. We have revised Fig. 2g to make the H-bonds more clearly (see below).\n8. Do the authors know whether the deltaTMD0-L0 construct is still folded properly either via SEC or mass photometry?\nResponse: Thanks for pointing this out. We have run SEC of this construct (see below). The protein is well-folded adjusted from the single peak in gel filtration.\n9. The authors are commended for attempts to separate monomers and dimers with different detergents. It seems for the wildtype protein that the different detergents change the proportion of monomer/dimer. Can the authors comment on that?\nResponse: As the reviewer pointed out, different detergents do have effects on the proportion of monomer/dimer of AtABCC2. As we observed in the dimer structure of AtABCC2, besides the interactions between TM12 and TM13 from the two protomers, lipid molecules also mediate the dimer formation. Changing the protein into different detergents may alter the binding of these lipid molecules to the protein during protein extraction, thus changing the proportion of monomer/dimer of AtABCC2.\n10. TMD2 in line 214 should be TMD1, as TM7 and TM8 are part of TMD1. This goes back to minor point #4 above.\nResponse: Thanks for pointing this out. We have renumbered the TMs in the revised manuscript according to the reviewer’s suggestion.\n11. In the dimeric ABCC2EQ structure, the orientation of the NBDs changes. Can the authors comment on why the catalytic mutation changes the orientation of the NBDs, either through their structures or comparison with other work?\nResponse: Thanks for pointing this out. Indeed, the E-to-Q mutation leads to movement of the NBDs closer together compared to the WT structure of AtABCC2. As we observed in the WT AtABCC2 structure, the map resolutions for the NBDs are relatively low, suggesting high structural flexibility of these NBD domains. But for the E-to-Q mutant, the map resolutions for the NBDs are better. It suggests that the E-to-Q mutation may alter the thermodynamics of ABCC2, shifting the transporter into such a more energy-favorable state different from the wild-type. And once ATP binds to the transporter, the NBDs move further together to form the occluded state. The structural dynamics of the NBDs in the ABC transporters have been investigated by different methods like structural determinations, single-molecule fluorescence spectroscopy, and molecular dynamics simulations. Notably, for the yeast homologue Ycf1, two distinct conformations were observed in the EQ mutant in the absence of ATP, both the inward-facing and the occluded conformations, further suggesting the intrinsic dynamics of NBDs (Khandelwal et al., Nature Communications, 2022.).\n12. Lines 317-319 describe where ATP binds at the NBDs in AtABCC2. The AtABCC2 ATP binding site is similar to that in other ABC transporters. The authors should state this and provide references.\nResponse: Thanks for the suggestion. We have added this point and related references in the revised manuscript (lines 321-324).\n13. The authors also state (319-321) that a mixture of salt bridges and H-bonds form between ATP and the NBDs, listing V738 as one of the NBD1 residues that binds ATP. A Val can not form H-bonds, unless the backbone is involved. This should be clarified.\nResponse: Thanks for pointing this out. Indeed, V738 forms hydrogen bond with ATP through its backbone carbonyl group. We have clarified this in the revised manuscript (lines 324-326).\n14. The protein interactions shown in Fig. S13 should have the associated density plotted as well.\nResponse: Thanks for the suggestion. We have included the density map in Fig. S13 (also see below).\n15. The transport cycle proposed for AtABCC2 in the discussion is similar to that proposed for other ABC proteins. Any similarities should be stated and referenced, and any differences could be highlighted.\nResponse: Thanks for the suggestion. This proposed cycle is similar to that proposed for other ABCC proteins. We have stated this point and added the references in the revised manuscript (lines 352-354)."}, {"round": 3, "reviewer_comments": {"reviewer_1": "All my concerns have been fully addressed in the revision. I have no further comments on the manuscript and fully support its publication.", "reviewer_2": "The authors are commended for the amount of work and for the work involved in revising the paper. However, there are some outstanding issues.\n1. I appreciate that the authors included the statistics for the Apo AtABCC2WT dimer and AtABCC2EQ+DNP-GS monomer structures in the Supplementary Table 1. I also appreciate the inclusion of the validation files for the structures. Edition the Supplementary Table 1 and including all validation reports addresses points 2 and 3 raised previously. However, I have some questions.\nFirst, the clash score of the validation report does not always match the value in Supplementary Table 1. For example, a clash score of 5.72 is reported in the table for the AtABCC2EQ+DMNP-GS monomer, but the validation report indicates a clash score of 7.\nAdditionally, a clash score of 4.28 is reported in the table for the AtABCC2EQ+DMNP-GS dimer, but the value in the validation report is 19. Why is this clash score so much larger than the rest of the structures?\nThe values in the validation report should be reflected in the table.\n2. I also appreciate that the authors added cryo-EM density when sidechains are shown for Fig. S6e and Fig. 3c. However, the figures do not adequately show the sidechain density, making it impossible to evaluate the fit of the model to the density. For The overlap of the sidechain densities with the models in Fig. 3c (left side showing interactions of TM helices across the dimer) and in Fig. S6e are difficult to evaluate. The authors could alleviate this problem by showing the sidechains and density in the top-down orientation, or perhaps by having a panel that only shows one protomer. The authors also haven’t labeled the residues depicted in these figures. This is important because the validation files indicate multiple positions where the model doesn’t fit in the map, which I assume is the region with lower resolution and not the region depicted in the figure. (Note that for the apo AtABCC2 monomer structure, shown in Fig. 2, the sidechain density is easy to see and the associated residues are clearly labeled. The same is true for the density added to Fig. S13, where residue labels are also shown.)\nDensity is missing for sidechains shown in Fig. 5e, Supplementary Fig. 11b (as is density for the substrate), and should be included.\nMinor points.\n1. The figure legend to Fig. 2g needs to be edited to indicate that the red lines indicate backbone H-bonds.\n2. The authors indicate in their rebuttal letter that they used SEC to show that the deltaTMD0+L0 construct is folded. However, this is not mentioned anywhere.\n3. The statement on lines 321-324 should be rewritten to indicate that binding of ATP and the observed dimerization of the NBDs is typical of what is seen in other ABC proteins. The way it is written at the moment suggests that only the sequences of the Walker A, Walker B, and signature motif are conserved."}, "author_response": "Response: We thank the reviewer for the positive feedback on our work and for the constructive and helpful comments. The raised points are addressed below:\n1. I appreciate that the authors included the statistics for the Apo AtABCC2WT dimer and AtABCC2EQ+DNP-GS monomer structures in the Supplementary Table 1. I also appreciate the inclusion of the validation files for the structures. Edition the Supplementary Table 1 and including all validation reports addresses points 2 and 3 raised previously. However, I have some questions.\nFirst, the clash score of the validation report does not always match the value in Supplementary Table 1. For example, a clash score of 5.72 is reported in the table for the AtABCC2EQ+DMNP-GS monomer, but the validation report indicates a clash score of 7.\nResponse: Thanks for pointing this out. We have further checked the coordinates and EM map, and updated the validation report for the AtABCC2 +DMNP-GS monomer. The EQ clash score value is 6.62 as reported after PHENIX refinement, which is now consistent with that value in the validation report (7).\nAdditionally, a clash score of 4.28 is reported in the table for the AtABCC2EQ+DMNP-GS dimer, but the value in the validation report is 19. Why is this clash score so much larger than the rest of the structures?\nResponse: Thanks for pointing this out. We have further checked the coordinates and EM map, and updated the validation report for the AtABCC2 +DMNP-GS dimer. Now the EQ clash score value is 4.21 as reported after PHENIX refinement, which is also consistent with that value in the validation report (4).\nThe values in the validation report should be reflected in the table.\nResponse: Thanks for pointing this out. We have further checked the values in the validation report and now they are consistent with those in the Supplementary Table 1, including the clashscores as mentioned above.\n2. I also appreciate that the authors added cryo-EM density when sidechains are shown for Fig. S6e and Fig. 3c. However, the figures do not adequately show the sidechain density, making it impossible to evaluate the fit of the model to the density. For The overlap of the sidechain densities with the models in Fig. 3c (left side showing interactions of TM helices across the dimer) and in Fig. S6e are difficult to evaluate. The authors could alleviate this problem by showing the sidechains and density in the top-down orientation, or perhaps by having a panel that only shows one protomer. The authors also haven’t labeled the residues depicted in these figures. This is important because the validation files indicate multiple positions where the model doesn’t fit in the map, which I assume is the region with lower resolution and not the region depicted in the figure. (Note that for the apo AtABCC2 monomer structure, shown in Fig. 2, the sidechain density is easy to see and the associated residues are clearly labeled. The same is true for the density added to Fig. S13, where residue labels are also shown.)\nDensity is missing for sidechains shown in Fig. 5e, Supplementary Fig. 11b (as is density for the substrate), and should be included.\nResponse: Thanks for the suggestion. Sorry for the unclear figures in the previous revision. As the review suggested, we have made a separate figure (Supplementary Fig. S9 of the revised manuscript, and also shown below) for the sidechain densities presented in Fig. 3c, Fig. 5e, and Fig. S11b. For Fig. S6e (now as Fig. S6f in the revised manuscript), we added an inset panel to show the EM densities for the sidechains presented in the transmembrane region. As suggested, we have also labeled the residues depicted in these figures.\nMinor points.\n1. The figure legend to Fig. 2g needs to be edited to indicate that the red lines indicate backbone H-bonds.\nResponse: Thanks for pointing this out. We have added this in the figure legend of Fig. 2g.\n2. The authors indicate in their rebuttal letter that they used SEC to show that the deltaTMD0+L0 construct is folded. However, this is not mentioned anywhere.\nResponse: Thanks for pointing this out. We have added this point and a figure showing the results in the revised manuscript (Supplementary Fig. S6b of the revised manuscript, and Page 8, Line 161-163).\n3. The statement on lines 321-324 should be rewritten to indicate that binding of ATP and the observed dimerization of the NBDs is typical of what is seen in other ABC proteins. The way it is written at the moment suggests that only the sequences of the Walker A, Walker B, and signature motif are conserved.\nResponse: Thanks for the suggestion. We have revised this statement following the reviewer’s suggestion (Page 16, Line 324-326)."}]

Decision letter
Reviewer #2:
Hello,
The authors have addressed all concerns.
One small thing they should do is indicate where the values in Supplementary Table 1 come from - PHENIX and not the PDB validation reports, as they indicate in the rebuttal letter.