Abstract
Bilirubin is mainly generated from the breakdown of heme when red blood cells reach the end of their lifespan. Accumulation of bilirubin in human body usually leads to various disorders, including jaundice and liver disease. Bilirubin is conjugated in hepatocytes and excreted to bile duct via the ATP-binding cassette transporter ABCC2, dysfunction of which would lead to Dubin-Johnson syndrome. Here we determine the structures of ABCC2 in the apo, substrate-bound and ATP/ADP-bound forms using the cryo-electron microscopy, exhibiting a full transporter with a regulatory (R) domain inserted between the two half modules. Combined with substrate-stimulated ATPase and transport activity assays, structural analysis enables us to figure out transport cycle of ABCC2 with the R domain adopting various conformations. At the rest state, the R domain binding to the translocation cavity functions as an affinity filter that allows the substrates of high affinity to be transported in priority. Upon substrate binding, the R domain is expelled from the cavity and docks to the lateral of transmembrane domain following ATP hydrolysis. Our findings provide structural insights into a transport mechanism of ABC transporters finely tuned by the R domain.