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Structures of ABCB4 provide insight into phosphatidylcholine translocation


Nosol, Kamil; Bang-Sørensen, Rose; Irobalieva, Rossitza N; Erramilli, Satchal K; Stieger, Bruno; Kossiakoff, Anthony A; Locher, Kaspar P (2021). Structures of ABCB4 provide insight into phosphatidylcholine translocation. Proceedings of the National Academy of Sciences of the United States of America, 118(33):e2106702118.

Abstract

ABCB4 is expressed in hepatocytes and translocates phosphatidylcholine into bile canaliculi. The mechanism of specific lipid recruitment from the canalicular membrane, which is essential to mitigate the cytotoxicity of bile salts, is poorly understood. We present cryogenic electron microscopy structures of human ABCB4 in three distinct functional conformations. An apo-inward structure reveals how phospholipid can be recruited from the inner leaflet of the membrane without flipping its orientation. An occluded structure reveals a single phospholipid molecule in a central cavity. Its choline moiety is stabilized by cation-π interactions with an essential tryptophan residue, rationalizing the specificity of ABCB4 for phosphatidylcholine. In an inhibitor-bound structure, a posaconazole molecule blocks phospholipids from reaching the central cavity. Using a proteoliposome-based translocation assay with fluorescently labeled phosphatidylcholine analogs, we recapitulated the substrate specificity of ABCB4 in vitro and confirmed the role of the key tryptophan residue. Our results provide a structural basis for understanding an essential translocation step in the generation of bile and its sensitivity to azole drugs.

Keywords: ABC transporter; cryo-EM; hepatocyte; membrane transport; phosphatidylcholine.

Abstract

ABCB4 is expressed in hepatocytes and translocates phosphatidylcholine into bile canaliculi. The mechanism of specific lipid recruitment from the canalicular membrane, which is essential to mitigate the cytotoxicity of bile salts, is poorly understood. We present cryogenic electron microscopy structures of human ABCB4 in three distinct functional conformations. An apo-inward structure reveals how phospholipid can be recruited from the inner leaflet of the membrane without flipping its orientation. An occluded structure reveals a single phospholipid molecule in a central cavity. Its choline moiety is stabilized by cation-π interactions with an essential tryptophan residue, rationalizing the specificity of ABCB4 for phosphatidylcholine. In an inhibitor-bound structure, a posaconazole molecule blocks phospholipids from reaching the central cavity. Using a proteoliposome-based translocation assay with fluorescently labeled phosphatidylcholine analogs, we recapitulated the substrate specificity of ABCB4 in vitro and confirmed the role of the key tryptophan residue. Our results provide a structural basis for understanding an essential translocation step in the generation of bile and its sensitivity to azole drugs.

Keywords: ABC transporter; cryo-EM; hepatocyte; membrane transport; phosphatidylcholine.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Clinic for Clinical Pharmacology and Toxicology
Dewey Decimal Classification:610 Medicine & health
Scopus Subject Areas:Health Sciences > Multidisciplinary
Uncontrolled Keywords:Multidisciplinary
Language:English
Date:17 August 2021
Deposited On:02 Feb 2022 18:19
Last Modified:26 Jun 2024 01:52
Publisher:National Academy of Sciences
ISSN:0027-8424
OA Status:Hybrid
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1073/pnas.2106702118
PubMed ID:34385322
Project Information:
  • : FunderSNSF
  • : Grant ID310030_189111
  • : Project TitleStructural and mechanistic studies of liver ABC transporters
  • Content: Accepted Version
  • Language: English