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

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...

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Published in:	Nature (London) Vol. 563; no. 7731; pp. 426 - 430
Main Authors:	Manolaridis, Ioannis, Jackson, Scott M., Taylor, Nicholas M. I., Kowal, Julia, Stahlberg, Henning, Locher, Kaspar P.
Format:	Journal Article
Language:	English
Published:	London Nature Publishing Group UK 01.11.2018 Nature Publishing Group
Subjects:	101/28 631/45/535/1258/1259 631/45/612/1237 631/535/1258/1259 82 ABC transporters Adenosine triphosphatase Adenosine triphosphate Adenosine Triphosphate - metabolism Antibodies ATP Binding Cassette Transporter, Subfamily G, Member 2 - chemistry ATP Binding Cassette Transporter, Subfamily G, Member 2 - genetics ATP Binding Cassette Transporter, Subfamily G, Member 2 - metabolism ATP Binding Cassette Transporter, Subfamily G, Member 2 - ultrastructure ATPases Binding Binding Sites Blood Brain Cancer Cancer research Cancer treatment Catalysis Cell membranes Cryoelectron Microscopy Cytoplasm Domains Electron microscopy Estrone Fetuses Glutamate Glutamine Humanities and Social Sciences Humans Hydrogen bonds Hydrophobicity Letter Leucine Ligands Microscopy Models, Molecular multidisciplinary Mutagenesis Mutant Proteins - chemistry Mutant Proteins - genetics Mutant Proteins - metabolism Mutant Proteins - ultrastructure Mutation Neoplasm Proteins - chemistry Neoplasm Proteins - genetics Neoplasm Proteins - metabolism Neoplasm Proteins - ultrastructure Pharmacokinetics Pharmacology Physiological aspects Protein Binding Protein Conformation Protein transport Proteins Recognition Science Science (multidisciplinary) Substrate inhibition Substrate Specificity Substrates Sulfates Symmetry Translocation Transmembrane domains Transport Transport proteins Xenobiotics United States > US Human ABCG2 Substrate-bound State Substrate-binding Cavity Nucleotide-binding Domain (NBD) External Cavity
ISSN:	0028-0836, 1476-4687, 1476-4687
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Summary:	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. Cryo-electron microscopy structures of the ABCG2 protein in ATP- and substrate-bound states reveal the location of substrate binding, conformational changes required for substrate translocation and how inhibitors might be distinguished from substrates.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 These authors contributed equally to this work
ISSN:	0028-0836 1476-4687 1476-4687
DOI:	10.1038/s41586-018-0680-3