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PreprintCited by 17Drug Transport and Resistance MechanismsAntibiotic Resistance in BacteriaATP Synthase and ATPases Research

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Structural basis for the transport and regulation mechanism of the Multidrug resistance-associated protein 2

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Koide, Eriko, Pietz, Harlan L., Beltran, Jean, Chen, Jue·2024·Nature Communications
DOI 10.1101/2024.06.24.600277PMC PMC11711199

Preprint — published as 10.1038/s41467-024-55810-w · Paper page

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Abstract

Multidrug resistance-associated protein 2 (MRP2) is an ATP-powered exporter important for maintaining liver homeostasis and a potential contributor to chemotherapeutic resistance. Deficiencies in MRP2 function are associated with Dubin-Johnson Syndrome and increased vulnerability to liver injury from cytotoxic drugs. Using cryogenic electron microscopy (cryo-EM), we determined the structures of human MRP2 in three conformational states: an autoinhibited state, a substrate-bound pre-translocation state, and an ATP-bound post-translocation state. These structures show that MRP2 functions through the classic alternating access model, driven by ATP binding and hydrolysis. Its cytosolic regulatory (R) domain serves as a selectivity gauge, wherein only sufficiently high concentrations of substrates can effectively compete with and disengage the R domain to initiate transport. Comparative structural analyses of MRP2 in complex with different substrates reveal how the transporter recognizes a diverse array of compounds, highlighting the transporter’s role in multidrug resistance.

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Figures

Fig. 1 The resting state of MRP2 reveals an auto-inhibitory R domain. A Domain organization of MRP2 with the secondary structure of the R domain depicted. Dashed lines indicate unresolved regions. B Ribbon representation of the structure of MRP2(E1462Q) in the inward-facing, ATP-bound conformation. Domains are colored as in (A). C Zoomed-in view with TMDs and NBDs represented as surfaces, colored as in (A) and (B), and the R domain in ribbon. Boxes indicate the positions of the two interfaces illustrated in panels (D and E). (D) R domain inserts into the substrate-binding site as viewed from the cytoplasm. Hydrogen bonds are indicated by red dashed lines. Colored as in (B). E An ATP molecule binds between the R domain (magenta) and NBD2 (blue). Cryo-EM density of ATP (contour level 0.08) is depicted as a tan transparent surface. Dashed lines indicate hydrogen bonds. F Relative ATPase activity of MRP2 variants. WT: wild type MRP2, EQ: the E1462Q variant, R928A/K932A: double mutation; and Scramble: a variant in which the R domain sequence is randomly scrambled. ATP hydrolysis rates were measured in the presence of 4 mM ATP/magnesium at a protein concentration of 75 nM, except for the E1462Q variant, which was 500 nM. Bar height represents the mean value of three separate measurements (unfilled dots) normalized to the average of WT activity. Statistical significance was calculated using the matched mixed-effects model and Dunnett’s multiple comparisons test on measured ATPase activity values (not normalized), comparing the mean of each variant to WT. The p value of MRP2(E1462Q) is 0.0011, for R928A/K932A is 0.0105, and for Scramble is < 0.0001 (*P < 0.05, **P < 0.005, ***P < 0.0001). G Relative ATPase activity of MRP2 WT and Scramble variants. Each datapoint represents the mean of 6 replicates, with ATPase activity normalized to the average activity at 4 mM ATP. Error bars represent standard deviation. The curve was fitted with the Michaelis-Menten least squares fit.
Fig. 1 The resting state of MRP2 reveals an auto-inhibitory R domain. A Domain organization of MRP2 with the secondary structure of the R domain depicted. Dashed lines indicate unresolved regions. B Ribbon representation of the structure of MRP2(E1462Q) in the inward-facing, ATP-bound conformation. Domains are colored as in (A). C Zoomed-in view with TMDs and NBDs represented as surfaces, colored as in (A) and (B), and the R domain in ribbon. Boxes indicate the positions of the two interfaces illustrated in panels (D and E). (D) R domain inserts into the substrate-binding site as viewed from the cytoplasm. Hydrogen bonds are indicated by red dashed lines. Colored as in (B). E An ATP molecule binds between the R domain (magenta) and NBD2 (blue). Cryo-EM density of ATP (contour level 0.08) is depicted as a tan transparent surface. Dashed lines indicate hydrogen bonds. F Relative ATPase activity of MRP2 variants. WT: wild type MRP2, EQ: the E1462Q variant, R928A/K932A: double mutation; and Scramble: a variant in which the R domain sequence is randomly scrambled. ATP hydrolysis rates were measured in the presence of 4 mM ATP/magnesium at a protein concentration of 75 nM, except for the E1462Q variant, which was 500 nM. Bar height represents the mean value of three separate measurements (unfilled dots) normalized to the average of WT activity. Statistical significance was calculated using the matched mixed-effects model and Dunnett’s multiple comparisons test on measured ATPase activity values (not normalized), comparing the mean of each variant to WT. The p value of MRP2(E1462Q) is 0.0011, for R928A/K932A is 0.0105, and for Scramble is < 0.0001 (*P < 0.05, **P < 0.005, ***P < 0.0001). G Relative ATPase activity of MRP2 WT and Scramble variants. Each datapoint represents the mean of 6 replicates, with ATPase activity normalized to the average activity at 4 mM ATP. Error bars represent standard deviation. The curve was fitted with the Michaelis-Menten least squares fit.CC BY · PubMed Central
Fig. 2 LTC4 binding releases R domain inhibition. A The overall structure of the LTC4-bound MRP2, colored as in Fig. 1B. B Superposition of the LTC4-bound structure, colored as in (A), with that of the substrate-free MRP2 (gray). Arrows indicate the inward movement upon LTC4 binding. C, D Local conformational changes at the LTC4-binding site, TMD1 in green, TMD2 in blue, LTC4 in tan, overlayed with inward-facing, substrate-free MRP2 (gray). Dashed red lines represent hydrogen bonding. E Molecular details of LTC4 binding MRP2, viewed from the cytosol. Colored as in (C), with TMD1 in green, TMD2 in blue, LTC4 in tan, and hydrogen bonds as red dashed lines. F ATP hydrolysis activity of MRP2 variants, normalized to the basal activity of wild type MRP2 at saturating ATP concentration. Each datapoint represents the mean value of 3 replicates. Error bars represent standard deviation. The curve was fitted with the quadratic binding formula for substrate binding (see “methods”). The Km values for LTC4: WT: 6.3 ± 1.5 μM, R928A/K932A: 11 ± 4 μM, and Scramble: 0.64 ± 0.28 μM (mean ± standard error).
Fig. 2 LTC4 binding releases R domain inhibition. A The overall structure of the LTC4-bound MRP2, colored as in Fig. 1B. B Superposition of the LTC4-bound structure, colored as in (A), with that of the substrate-free MRP2 (gray). Arrows indicate the inward movement upon LTC4 binding. C, D Local conformational changes at the LTC4-binding site, TMD1 in green, TMD2 in blue, LTC4 in tan, overlayed with inward-facing, substrate-free MRP2 (gray). Dashed red lines represent hydrogen bonding. E Molecular details of LTC4 binding MRP2, viewed from the cytosol. Colored as in (C), with TMD1 in green, TMD2 in blue, LTC4 in tan, and hydrogen bonds as red dashed lines. F ATP hydrolysis activity of MRP2 variants, normalized to the basal activity of wild type MRP2 at saturating ATP concentration. Each datapoint represents the mean value of 3 replicates. Error bars represent standard deviation. The curve was fitted with the quadratic binding formula for substrate binding (see “methods”). The Km values for LTC4: WT: 6.3 ± 1.5 μM, R928A/K932A: 11 ± 4 μM, and Scramble: 0.64 ± 0.28 μM (mean ± standard error).CC BY · PubMed Central
Fig. 3 The NBD-dimerized, outward-facing conformation. A Ribbon representation of the overall structure, colored as in Fig. 1B. Also shown is the cryo-EM density corresponding to the R domain at a contour level of 0.095 (magenta surface). B Zoomed-in view of the R domain, colored as in (A). Hydrogen bonds are represented as dashed lines. C, D conformational changes at the substrate-binding site in the inward-facing, LTC4-bound state (C) versus the outward-facing conformation (D). Residues in green correspond to TMD1, residues in blue correspond to TMD2. E The structure of the substrate-binding site in LTC4-bound conformation, viewed from the extracellular space. TMD1 depicted in green, TMD2 depicted in blue. Hydrogen bonds are represented as dashed lines. F The structure of the substrate-binding site in outward-facing conformation, viewed from the extracellular space.
Fig. 3 The NBD-dimerized, outward-facing conformation. A Ribbon representation of the overall structure, colored as in Fig. 1B. Also shown is the cryo-EM density corresponding to the R domain at a contour level of 0.095 (magenta surface). B Zoomed-in view of the R domain, colored as in (A). Hydrogen bonds are represented as dashed lines. C, D conformational changes at the substrate-binding site in the inward-facing, LTC4-bound state (C) versus the outward-facing conformation (D). Residues in green correspond to TMD1, residues in blue correspond to TMD2. E The structure of the substrate-binding site in LTC4-bound conformation, viewed from the extracellular space. TMD1 depicted in green, TMD2 depicted in blue. Hydrogen bonds are represented as dashed lines. F The structure of the substrate-binding site in outward-facing conformation, viewed from the extracellular space.CC BY · PubMed Central
Fig. 4 The MRP2 transport cycle. A In the absence of substrate, MRP2 rests in an auto-inhibited state. Colored as in Fig. 1B. Dotted magenta lines represent unresolved regions of R domain. In this state, ATP-binding does not induce NBD dimerization, but rather stabilizes R domain in the auto-inhibiting position. B Substrate displaces R domain from the auto-inhibited conformation. C Substrate is released in the NBD-dimerized conformation.
Fig. 4 The MRP2 transport cycle. A In the absence of substrate, MRP2 rests in an auto-inhibited state. Colored as in Fig. 1B. Dotted magenta lines represent unresolved regions of R domain. In this state, ATP-binding does not induce NBD dimerization, but rather stabilizes R domain in the auto-inhibiting position. B Substrate displaces R domain from the auto-inhibited conformation. C Substrate is released in the NBD-dimerized conformation.CC BY · PubMed Central
Fig. 5 Structural basis of multi-substrate recognition. A The structure of the substrate binding site observed in the LTC4-bound conformation (yellow). Residues in the P-pocket that interact with LTC4 are indicated. Hydrogen bonds are depicted as dotted red lines. B Schematic drawing of the LTC4 binding site. LTC4 is shown in burgundy, with unresolved hydrophobic tail in gray. Hydrogen bonds are depicted as dashed lines. C The structure of substrate-binding site observed in the BDT/cholesterol (CLR)-bound conformation (pink)29. BDT is depicted in tan, CLR in gray. D Schematic drawing of the interactions of BDT (burgundy) and CLR (black) with MRP2 as in (B). E The structure of binding site observed in the Probenecid/cholesterol-bound rMRP2 (blue)30. Probenecid is depicted in tan, cholesterol in gray. F Schematic drawing of the rMRP2 bound with probenecid (burgundy) and CHS (black). Portion of CHS which extends away from the substrate binding site residues is not pictured. In panels (B, D, and F) the conserved residues that form hydrogen bonds with substrates are highlighted in purple.
Fig. 5 Structural basis of multi-substrate recognition. A The structure of the substrate binding site observed in the LTC4-bound conformation (yellow). Residues in the P-pocket that interact with LTC4 are indicated. Hydrogen bonds are depicted as dotted red lines. B Schematic drawing of the LTC4 binding site. LTC4 is shown in burgundy, with unresolved hydrophobic tail in gray. Hydrogen bonds are depicted as dashed lines. C The structure of substrate-binding site observed in the BDT/cholesterol (CLR)-bound conformation (pink)29. BDT is depicted in tan, CLR in gray. D Schematic drawing of the interactions of BDT (burgundy) and CLR (black) with MRP2 as in (B). E The structure of binding site observed in the Probenecid/cholesterol-bound rMRP2 (blue)30. Probenecid is depicted in tan, cholesterol in gray. F Schematic drawing of the rMRP2 bound with probenecid (burgundy) and CHS (black). Portion of CHS which extends away from the substrate binding site residues is not pictured. In panels (B, D, and F) the conserved residues that form hydrogen bonds with substrates are highlighted in purple.CC BY · PubMed Central

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Metadata

Authors
Koide, E., Pietz, H. L., Beltran, J., & Chen, J.
Affiliations
  • Rockefeller University (US)
  • Davidson College (US)
  • Howard Hughes Medical Institute (US)
Corresponding author
Chen, Jue
ORCID
Venue
Nature Communications
Publisher
openRxiv
Published
2024-06-25
Volume
16
Issue
1
Pages
484-484
ISSN
2041-1723 (ISSN-L)
DOI
10.1101/2024.06.24.600277
PMC
PMC11711199
OpenAlex
W4406152854
Cited by
17
References
20
Citations by year
2026: 11 · 2025: 5 · 2024: 1
FWCI
6.59
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Howard Hughes Medical Institute
  • Weill Cornell Medical College (T32GM007739)
  • National Cancer Institute (F30CA257282)
  • National Institute of General Medical Sciences (T32GM007739)
Topics
  • Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
  • Health Sciences › Medicine › Microbiology (medical) › Antibiotic Resistance in Bacteria
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › ATP Synthase and ATPases Research
Keywords
MRP2, ABC transporters, ATP-binding cassette, substrate specificity, regulatory domain, autoinhibition, conformational change, nucleotide-binding domain, substrate binding, multidrug resistance, chemoresistance, liver homeostasis, drug efflux

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Koide, E., Pietz, H. L., Beltran, J., & Chen, J. (2024). Structural basis for the transport and regulation mechanism of the Multidrug resistance-associated protein 2. Nature Communications. https://doi.org/10.1101/2024.06.24.600277

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