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Cited by 202Drug Transport and Resistance MechanismsAntibiotic Resistance in BacteriaProtein Structure and Dynamics

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ABCG2 transports anticancer drugs via a closed-to-open switch

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Orlando, Benjamin J., Liao, Maofu·2020·Nature Communications
DOI 10.1038/s41467-020-16155-2PMC PMC7210939
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Abstract

ABCG2 is an ABC transporter that extrudes a variety of compounds from cells, and presents an obstacle in treating chemotherapy-resistant cancers. Despite recent structural insights, no anticancer drug bound to ABCG2 has been resolved, and the mechanisms of multidrug transport remain obscure. Such a gap of knowledge limits the development of novel compounds that block or evade this critical molecular pump. Here we present single-particle cryo-EM studies of ABCG2 in the apo state, and bound to the three structurally distinct chemotherapeutics. Without the binding of conformation-selective antibody fragments or inhibitors, the resting ABCG2 adopts a closed conformation. Our cryo-EM, biochemical, and functional analyses reveal the binding mode of three chemotherapeutic compounds, demonstrate how these molecules open the closed conformation of the transporter, and establish that imatinib is particularly effective in stabilizing the inward facing conformation of ABCG2. Together these studies reveal the previously unrecognized conformational cycle of ABCG2.

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Fig. 1 Cryo-EM structure of apo-closed ABCG2. a Final cryo-EM map of nanodisc reconstituted ABCG2 in the absence of added ligands. Nanodisc density is shown in white, and TM5 is colored green. In the apo-closed conformation the TM helices are closed, while the NBDs remain open in a nucleotide-free state. b Overall arrangement of the TM helices in the apo-closed conformation. TM2 is colored magenta, with the central portion that unravels colored cyan. TM5 is colored green. c View from the extracellular space of the TM helix arrangement in the apo-closed conformation. In the apo-closed conformation, Phe439 (cyan sticks) is flipped outward away from the center of the transporter, and rotation of TM5 causes Phe545 (orange sticks) to point towards the opposite ABCG2 monomer, and brings several sulfur containing sidechains (blue and yellow sticks) to the central dimer axis. d View from the extracellular space of the TM helix arrangement in previously determined inward facing ABCG2 cryo-EM structures. TM helices and residues are colored the same as in (c). Red arrows indicate the direction of TM5 rotation in going from inward facing to apo-closed states.
Fig. 1 Cryo-EM structure of apo-closed ABCG2. a Final cryo-EM map of nanodisc reconstituted ABCG2 in the absence of added ligands. Nanodisc density is shown in white, and TM5 is colored green. In the apo-closed conformation the TM helices are closed, while the NBDs remain open in a nucleotide-free state. b Overall arrangement of the TM helices in the apo-closed conformation. TM2 is colored magenta, with the central portion that unravels colored cyan. TM5 is colored green. c View from the extracellular space of the TM helix arrangement in the apo-closed conformation. In the apo-closed conformation, Phe439 (cyan sticks) is flipped outward away from the center of the transporter, and rotation of TM5 causes Phe545 (orange sticks) to point towards the opposite ABCG2 monomer, and brings several sulfur containing sidechains (blue and yellow sticks) to the central dimer axis. d View from the extracellular space of the TM helix arrangement in previously determined inward facing ABCG2 cryo-EM structures. TM helices and residues are colored the same as in (c). Red arrows indicate the direction of TM5 rotation in going from inward facing to apo-closed states.CC BY · PubMed Central
Fig. 2 Disulfide crosslinking of ABCG2 in different conformational states. a View from the extracellular space of ABCG2 TM helices in the ATP bound state (PDBid 6HZM). A cysteine was introduced at position 534 to probe disulfide crosslinking across the dimer interface. b Non-reducing SDS-PAGE with in-gel GFP fluorescence to monitor oxidant-induced crosslinking of the Val534Cys mutant. Upper band represents disulfide crosslinked ABCG2. c Densitometry quantification of crosslinking in (b). Data represents the average of three independent experiments with SEM. d View from the extracellular space of ABCG2 TM helices in the apo-closed state. A cysteine was introduced at position 537 to probe disulfide crosslinking across the dimer interface. e Non-reducing SDS-PAGE with in-gel GFP fluorescence to monitor oxidant-induced crosslinking of the Val534Cys mutant. Upper band represents disulfide crosslinked ABCG2. f Densitometry quantification of crosslinking in (e). Data represents the average of three independent experiments with SEM. Source data are provided as a Source data file.
Fig. 2 Disulfide crosslinking of ABCG2 in different conformational states. a View from the extracellular space of ABCG2 TM helices in the ATP bound state (PDBid 6HZM). A cysteine was introduced at position 534 to probe disulfide crosslinking across the dimer interface. b Non-reducing SDS-PAGE with in-gel GFP fluorescence to monitor oxidant-induced crosslinking of the Val534Cys mutant. Upper band represents disulfide crosslinked ABCG2. c Densitometry quantification of crosslinking in (b). Data represents the average of three independent experiments with SEM. d View from the extracellular space of ABCG2 TM helices in the apo-closed state. A cysteine was introduced at position 537 to probe disulfide crosslinking across the dimer interface. e Non-reducing SDS-PAGE with in-gel GFP fluorescence to monitor oxidant-induced crosslinking of the Val534Cys mutant. Upper band represents disulfide crosslinked ABCG2. f Densitometry quantification of crosslinking in (e). Data represents the average of three independent experiments with SEM. Source data are provided as a Source data file.CC BY · PubMed Central
Fig. 3 Imatinib traps ABCG2 in an inward conformation. a Cryo-EM map of ABCG2 bound to imatinib. ABCG2 is in an inward facing conformation. TM5 is colored green, and imatinib is colored orange. Red dashed box indicates the position of bound imatinib. b Zoomed-in and rotated view of the highlighted area in (a) showing the cryo-EM map for imatinib. c View of the imatinib binding pocket from the extracellular space. Imatinib binds on the 2-fold axis between ABCG2 monomers and is sandwiched between Phe439 sidechains from each monomer.
Fig. 3 Imatinib traps ABCG2 in an inward conformation. a Cryo-EM map of ABCG2 bound to imatinib. ABCG2 is in an inward facing conformation. TM5 is colored green, and imatinib is colored orange. Red dashed box indicates the position of bound imatinib. b Zoomed-in and rotated view of the highlighted area in (a) showing the cryo-EM map for imatinib. c View of the imatinib binding pocket from the extracellular space. Imatinib binds on the 2-fold axis between ABCG2 monomers and is sandwiched between Phe439 sidechains from each monomer.CC BY · PubMed Central
Fig. 4 Binding of MXN and SN38 to ABCG2. a View of ABCG2 TM helices in the inward conformation bound to MXN. TM2 is colored magenta, and TM5 is colored green. b View from the extracellular space showing that MXN binds between Phe439 from opposing monomers. c Zoomed-in view of the MXN binding site with MXN shown in blue. TM2 and associated density from each monomer is shown as magenta sticks. d View of ABCG2 TM helices in the inward conformation bound to SN38. TM2 is colored magenta, and TM5 is colored green. e View from the extracellular space showing that SN38 binds between Phe439 from opposing monomers. f Zoomed-in view of the SN38 binding site with SN38 shown as yellow sticks. TM2 is shown as magenta sticks.
Fig. 4 Binding of MXN and SN38 to ABCG2. a View of ABCG2 TM helices in the inward conformation bound to MXN. TM2 is colored magenta, and TM5 is colored green. b View from the extracellular space showing that MXN binds between Phe439 from opposing monomers. c Zoomed-in view of the MXN binding site with MXN shown in blue. TM2 and associated density from each monomer is shown as magenta sticks. d View of ABCG2 TM helices in the inward conformation bound to SN38. TM2 is colored magenta, and TM5 is colored green. e View from the extracellular space showing that SN38 binds between Phe439 from opposing monomers. f Zoomed-in view of the SN38 binding site with SN38 shown as yellow sticks. TM2 is shown as magenta sticks.CC BY · PubMed Central
Fig. 5 Model of drug capture and extrusion by ABCG2. In the absence of ligands ABCG2 adopts an apo-closed conformation (1). In this conformation the NBDs are well separated, TM5 (green/yellow bar) is shifted towards the dimer axis, and TM2 (purple spring) is unwound near the center of the helix. Binding of substrates and inhibitors shifts ABCG2 to the inward facing conformation (2). In this conformation TM2 regains helical character, TM5 moves laterally to the sides of the transporter and rotates away from the dimer axis. Bound drug molecules are captured between Phe439 from each monomer (cyan circle). Subsequent binding of ATP in the NBDs causes the TM helix bundles to collapse toward one another, pushing the bound drug molecule through the leucine plug (red flipper) and into the extracellular space. ATP hydrolysis resets the transporter to the apo-closed conformation.
Fig. 5 Model of drug capture and extrusion by ABCG2. In the absence of ligands ABCG2 adopts an apo-closed conformation (1). In this conformation the NBDs are well separated, TM5 (green/yellow bar) is shifted towards the dimer axis, and TM2 (purple spring) is unwound near the center of the helix. Binding of substrates and inhibitors shifts ABCG2 to the inward facing conformation (2). In this conformation TM2 regains helical character, TM5 moves laterally to the sides of the transporter and rotates away from the dimer axis. Bound drug molecules are captured between Phe439 from each monomer (cyan circle). Subsequent binding of ATP in the NBDs causes the TM helix bundles to collapse toward one another, pushing the bound drug molecule through the leucine plug (red flipper) and into the extracellular space. ATP hydrolysis resets the transporter to the apo-closed conformation.CC BY · PubMed Central

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Metadata

Authors
Orlando, B. J., & Liao, M.
Affiliations
Harvard University (US)
Corresponding author
Liao, Maofu
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2020-05-08
Volume
11
Issue
1
Pages
2264-2264
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-020-16155-2
PMC
PMC7210939
OpenAlex
W3021390585
Cited by
198
References
57
Citations by year
2026: 20 · 2025: 18 · 2024: 31 · 2023: 39 · 2022: 31 · 2021: 46 · 2020: 12
FWCI
11.92
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
American Cancer Society (PF-17-212-01–DMC)
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 › Protein Structure and Dynamics
Keywords
ABCG2, ABC transporters, multidrug resistance, anticancer drug transport, conformational cycle, imatinib, inward-facing conformation, drug efflux
MeSH terms
Imatinib Mesylate, ATP Binding Cassette Transporter, Subfamily G, Member 2, Antineoplastic Agents, Biological Transport, Disulfides, Humans, Ligands, Mitoxantrone, Models, Biological, Protein Structure, Secondary, HEK293 Cells

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