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

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ABCG2 is a multidrug ATP-binding cassette transporter expressed in the plasma membranes of various tissues and tissue barrier [1–4]. It translocates endogenous substrates, affects the pharmacokinetics of many drugs, and has a protective role 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 small-molecule and antibody inhibition [13, 14], but the mechanism of substrate recognition and ATP-driven transport are currently unknown. Here we present high-resolution cryo-EM structures of human ABCG2 in two key states, a substrate-bound pre-translocation state and an ATP-bound post-translocation state. For both structures, a mutant containing a glutamine replacing the catalytic glutamate (ABCG2EQ) was used, 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-sulphate (E1S) is bound in a central, hydrophobic, and cytoplasm-facing cavity about halfway across the membrane. Only one molecule of E1S can bind in the observed binding mode. In the ATP-boundstate, the substrate-binding cavity has completely 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 (TMDs), pivoting of the nucleotide-binding domains (NBDs), and a change in the relative orientation of the NBD subdomains. Mutagenesis of residues contacting bound E1S or in the translocation pathway, followed by in vitro characterization of transport and ATPase activities, demonstrated their roles in substrate recognition and revealed the importance of a leucine residue forming a ‘plug’ between the two cavities. Our results reveal how ABCG2 harnesses the energy of ATP binding to extrude E1S and other substrates and suggest that the size and binding affinity of compounds are important parameters in distinguishing substrates from inhibitors.