Structures of ABCG2 under turnover conditions reveal a key step in drug transport mechanism

ABCG2 is a multidrug transporter expressed widely in the human body. Its physiological substrates include steroid derivatives and uric acid. In addition, it extrudes many structurally diverse cytotoxic drugs from various cells, thus affecting drug pharmacokinetics and contributing to multidrug resistance of cancer cells. Previous studies have revealed structures of ABCG2 bound to transport substrates, nucleotides, small-molecule inhibitors and inhibitory antibodies. However, the transport mechanism is not well-understood because all previous structures described trapped states, where the reaction cycle was halted by the absence of substrates or ATP, mutation of catalytic residues, or the presence of inhibitors. Here we present cryo-EM structures of nanodisc-reconstituted human ABCG2 under turnover conditions containing either the endogenous substrate estrone-3-sulfate or the exogenous substrate topotecan. We found two distinct conformational states in which both the transport substrates and ATP are bound. Whereas the state turnover-1 features more widely separated NBDs and an accessible cavity between the TMDs, turnover-2 features semi-closed NBDs and an almost fully occluded cavity between the TMDs. The transition from turnover-1 to turnover-2 includes conformational changes that link the binding of ATP by the NBDs to the closing of the cytoplasmic side of the TMDs. The size of the substrate appears to control which turnover state corresponds to the main state in the transport cycle. The transition from turnover-1 to turnover-2 is the likely bottleneck or rate-limiting step of the reaction cycle, where the discrimination of substrates and inhibitors occurs. Our results provide a structural basis of substrate specificity of ABCG2 and provide key insight to understand the transport cycle.