title: Attachment of a 'molecular spring' restores drug-stimulated ATPase activity to P-glycoprotein lacking both Q loop glutamines.
abstract: P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump that is clinically important because it confers multidrug resistance. Drugs bind at the interface between the transmembrane domains to activate ATPase activity at the two nucleotide-binding domains (NBDs). Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. Recently, it was reported that the conserved glutamines residues (Gln475 in NBD1 and Gln1118 in NBD2) in the Q loops of P-gp when mutated to alanine completely inhibited the drug-stimulated ATPase activity. It is unknown why the glutamine residues (Gln475 and Gln1118) in the Q loops of the NBDs of P-gp are required for drug-stimulated ATPase activity. Here we show that introduction of these mutations into the L175C/N820C mutant (L175C/N820C/Q475A/Q1118A) also abolished drug-stimulated ATPase activity. The ATPase activity was restored however, when the L175C/N820C/Q475A/Q1118A mutant was cross-linked with a flexible disulfide cross-linker. These results suggest that both Q-loop glutamines are not required for ATP hydrolysis and they might function as part of a spring-like mechanism in facilitating the open (inactive) to closed (active) conformational change during ATP hydrolysis. The molecular spring-like action of the Q-loop glutamines during drug-stimulated ATPase activity is likely mimicked by the attachment of the flexible cross-linker.
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: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004351:Drug Resistance; D018432:Drug Resistance, Multiple; D005973:Glutamine / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D008055:Lipids / Q000737:chemistry; D008968:Molecular Conformation; D009154:Mutation; D011487:Protein Conformation; D000072417:Protein Domains
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D003432:Cross-Linking Reagents; D003902:Detergents; D004220:Disulfides; D008055:Lipids; D005973:Glutamine; D000251:Adenosine Triphosphatases
keywords: ABC protein; Disulfide cross-linking; Drug-stimulated ATPase activity; Molecular spring; P-glycoprotein