ABCG2/BCRP transport mechanism revealed through kinetically excited targeted molecular dynamics simulations

ABCG2/BCRP is an ABC transporter that plays an important role in tissue protection by exporting endogenous substrates and xenobiotics. ABCG2 is of major interest due to its involvement in multidrug resistance (MDR), and understanding its complex efflux mechanism is essential to preventing MDR and drug-drug interactions (DDI). ABCG2 export is characterized by two major conformational transitions between inward- and outward-facing states, the structures of which have been resolved. Yet, the entire transport cycle has not been characterized to date. Our study bridges the gap between the two extreme conformations by studying connecting pathways. We developed an innovative approach to enhance molecular dynamics simulations, ‘kinetically excited targeted molecular dynamics’, and successfully simulated the transitions between inward- and outward-facing states in both directions and the transport of the endogenous substrate estrone 3-sulfate. We discovered an additional pocket between the two substrate-binding cavities and found that the presence of the substrate in the first cavity is essential to couple the movements between the nucleotide-binding and transmembrane domains. Our study shed new light on the complex efflux mechanism, and we provided transition pathways that can help to identify novel substrates and inhibitors of ABCG2 and probe new drug candidates for MDR and DDI.