A dual sensor regulates P-glycoprotein's structural plasticity

P-glycoprotein is an efflux pump with an exceptionally broad substrate profile which drives its profound clinical impact. Despite its biological importance, it remains obscure how P-glycoprotein achieves its polyspecificity and how substrate binding and the lipid environment stimulate its activity. Structural data highlight the importance of transmembrane helices 4 and 10, which surround the binding pocket, and identify them as key players in substrate recognition. Here, we used cryo-EM to study P-glycoprotein in detergent and nanodiscs to strategically leverage environment- and substrate-dependent phenotypes. This approach allowed us to decipher unexpected and distinct roles of transmembrane helices 4 and 10, which structurally explain differences in ATPase activity. Our data highlights helix 4 as an environment sensor and helix 10 as the key player in substrate recognition constituting a dual regulation mechanism for the functional plasticity of P-glycoprotein, and visualizes the intricate interplay between a membrane protein and its environment.