title: P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface.
abstract: P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids.
authors.affiliation: Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
authors.forename: Tip W
authors.initials: TW
authors.lastname: Loo
authors.affiliation: Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada. Electronic address: david.clarke@utoronto.ca.
authors.forename: David M
authors.initials: DM
authors.lastname: Clarke
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000648:ultrastructure*; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000648:ultrastructure*; D001665:Binding Sites; D003902:Detergents / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008956:Models, Chemical; D062105:Molecular Docking Simulation; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003902:Detergents; D008055:Lipids; D000251:Adenosine Triphosphatases
keywords: ABC protein; ATPase activity; Disulfide cross-linking; P-glycoprotein; Switch; Transmission interface