Direct Observation of Unidirectional Cholesterol Flipping on the Surface of P-Glycoprotein

Abstract

P-glycoprotein (Pgp) is a biomedically important member of the largest superfamily of ABC transporters and mediates multidrug resistance in many cancer types. Substrate binding and transport in Pgp are modulated by the presence of cholesterol in the membrane, while cholesterol itself is a substrate and exported by Pgp. Structural information on cholesterol binding sites and mechanistic details of its transport pathway are largely unknown. In this study, a set of 35 independent simulations of Pgp oriented independently in cholesterol-rich lipid bilayers were performed, adding to a total of 7 μs, enabling extensive sampling of lipid-protein interactions. From the ensemble of cholesterol molecules (∼4700) sampled in these simulations, one complete and two partial cholesterol flipping events were captured. All of the flipping events visited a region formed by transmembrane (TM) helices TM1, TM2, and TM11 with Tyr 49 and Tyr 126 as key residues interacting with cholesterol. This region has been identified with two cholesterol-recognition amino acid sequence (CRAC) motifs. Cholesterol flipping from the inner leaflet initially binds to this motif and assumes a 90° angle to the membrane normal burying its only hydroxyl group within the surface of the protein followed by flipping to the outer leaflet. Convergence and clustering analyses of cholesterol molecules around Pgp revealed that cholesterol binds to specific regions formed between TM1-6 and TM8 helices interacting through its rough surface. From the generated ensemble of cholesterol sampling around Pgp, only a single complete cholesterol flipping from inner leaflet to outer leaflet has been directly observed, which supports the known flippase activity of Pgp. Our study is the first to report that substrate translocation by Pgp might take place through pathways on the surface of the protein.