The Human ABCG2 Transporter Relies on a Triple-Gated Translocation Channel for Multidrug Efflux

The human ABCG2 drug transporter plays essential roles in physiological detoxification across barrier tissues and in anticancer multidrug resistance. However, the routes for drug extrusion and gating mechanisms remain elusive. Here, we demonstrate that the inner core of the ABCG2 structure holds two cavities that are delimited by three gates, revealing a possible substrate translocation channel within the transporter core. Drugs can enter via an intracellular entry gate to become trapped in the voluminous central cavity. The cavity is surrounded by a cluster of three conserved phenylalanines providing aromatic rings for a “clamp-push-seal” motion to ensure unidirectional substrate movement towards the upper cavity. Two ABCG2-unique residues, T435 and N436, are the critical discriminators for substrate selectivity prior moving through the valve gate. The dynamic outer cavity is covered by the extracellular lid architecture functioning as the final gate to mediate drug extrusion. This work unravels unprecedented mechanistic details about critical gating steps, including the order of events that drive ABCG2-mediated drug efflux.