Attachment of a 'molecular spring' restores drug-stimulated ATPase activity to P-glycoprotein lacking both Q loop glutamines.

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.