Structural Switches Regulating the Alternating Access Mechanism of Human P-Glycoprotein

Abstract

P-glycoprotein (Pgp) is a biomedically important ATP-binding cassette (ABC) transporter involved in the development of multidrug resistance in cancer cells. In spite of the canonized work on the transporter function, a lack of general consensus remains concerning the structural details of the different conformational states of Pgp. A major limitation in the study of conformational transitions in ABC transporters is that the process is driven by formation of specific ATP interactions with the nucleotide binding domains (NBDs) of the transporter. Using a novel steered molecular dynamics methodology coupling specific ligand-protein and inter-domain salt-bridge interactions with global conformational changes, we successfully generate four different states of human Pgp, well-separated on the conformational landscape. These include the first stable and catalytically active ATP-bound outward-facing (OF) conformation displaying an open extracellular gate stabilized by lipid binding inside the lumen, as well as an APO (nucleotide-free) inward-facing (IF) conformation showing an open cytoplasmic gate and a large central drug-binding cavity capable of recruiting substrates. In addition, we characterize the only available ATP-bound human Pgp structure as the substrate-free occluded (OC) state of the transporter, with the lumen present in a collapsed state, and likely responsible for the basal-ATPase activity of Pgp. Binding of ATP to the APO IF conformation promotes the closure of the cytoplasmic gate as well as the collapse of the central cavity, likely inducing the transition to the substrate-free OC state. Differential formation of specific salt bridges located at the drug-entry portals in the transmembrane domain as well as between the two NBDs may play an important role in allowing transition between the ATP-bound IF and OF states. The study provides key molecular insights into the specific local switches regulating the global conformational transitions in the transport cycle of human Pgp.