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Cryo-EM structures of a human ABCG2 mutant trapped in ATP-bound and substrate-bound states

Manolaridis, Ioannis and Jackson, Scott M. and Taylor, Nicholas M. I. and Kowal, Julia and Stahlberg, Henning and Locher, Kaspar P.. (2018) Cryo-EM structures of a human ABCG2 mutant trapped in ATP-bound and substrate-bound states. Nature, 563 (7731). pp. 426-430.

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Official URL: https://edoc.unibas.ch/66722/

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Abstract

ABCG2 is a transporter protein of the ATP-binding-cassette (ABC) family that is expressed in the plasma membrane in cells of various tissues and tissue barriers, including the blood-brain, blood-testis and maternal-fetal barriers; 1-4; . Powered by ATP, it translocates endogenous substrates, affects the pharmacokinetics of many drugs and protects against a wide array of xenobiotics, including anti-cancer drugs; 5-12; . Previous studies have revealed the architecture of ABCG2 and the structural basis of its inhibition by small molecules and antibodies; 13,14; . However, the mechanisms of substrate recognition and ATP-driven transport are unknown. Here we present high-resolution cryo-electron microscopy (cryo-EM) structures of human ABCG2 in a substrate-bound pre-translocation state and an ATP-bound post-translocation state. For both structures, we used a mutant containing a glutamine replacing the catalytic glutamate (ABCG2; EQ; ), which resulted in reduced ATPase and transport rates and facilitated conformational trapping for structural studies. In the substrate-bound state, a single molecule of estrone-3-sulfate (E; 1; S) is bound in a central, hydrophobic and cytoplasm-facing cavity about halfway across the membrane. Only one molecule of E; 1; S can bind in the observed binding mode. In the ATP-bound state, the substrate-binding cavity has collapsed while an external cavity has opened to the extracellular side of the membrane. The ATP-induced conformational changes include rigid-body shifts of the transmembrane domains, pivoting of the nucleotide-binding domains (NBDs), and a change in the relative orientation of the NBD subdomains. Mutagenesis and in vitro characterization of transport and ATPase activities demonstrate the roles of specific residues in substrate recognition, including a leucine residue that forms a 'plug' between the two cavities. Our results show how ABCG2 harnesses the energy of ATP binding to extrude E; 1; S and other substrates, and suggest that the size and binding affinity of compounds are important for distinguishing substrates from inhibitors.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics
05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Stahlberg)
UniBasel Contributors:Stahlberg, Henning
Item Type:Article, refereed
Article Subtype:Research Article
ISSN:1476-4687
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:26 Apr 2020 19:15
Deposited On:26 Apr 2020 19:15

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