Properties of P-glycoprotein with mutations in the "catalytic carboxylate" glutamate residues.

It is known from earlier work that two conserved Glu residues, designated "catalytic carboxylates," are critical for function in P-glycoprotein (Pgp). Here the role of these residues (Glu-552 and Glu-1197 in mouse MDR3 Pgp) was studied further. Mutation E552Q or E1197Q reduced Pgp-ATPase to low but still measurable rates.

Synergy between conserved ABC signature Ser residues in P-glycoprotein catalysis.

Functional roles of the two ABC signature sequences ("LSGGQ") in the N- and C-terminal nucleotide binding domains of P-glycoprotein were studied by mutating the conserved Ser residues to Ala. The two single mutants (S528A; S1173A) each impaired ATPase activity mildly, and showed generally symmetrical effects on function, consistent with equivalent mechanistic roles of the two nucleotide sites. Synergy between the two mutations when combined was remarkable and resulted in strong catalytic impairment.

Purification and characterization of N-glycosylation mutant mouse and human P-glycoproteins expressed in Pichia pastoris cells.

P-glycoprotein confers multidrug resistance in mammalian cells and basic structure-function studies of it are germane to anti-cancer and anti-AIDS therapy. Pure, detergent-soluble mouse MDR3 and human MDR1 P-glycoproteins have recently been obtained in sufficient quantity for high-resolution structure analysis after expression in Pichia pastoris (N. Lerner-Marmarosh et al.

Cysteines 431 and 1074 are responsible for inhibitory disulfide cross-linking between the two nucleotide-binding sites in human P-glycoprotein.

Human wild-type and Cys-less P-glycoproteins were expressed in Pichia pastoris and purified in high yield in detergent-soluble form. Both ran on SDS gels as a single 140-kDa band in the presence of reducing agent and showed strong verapamil-stimulated ATPase activity in the presence of added lipid. The wild type showed spontaneous formation of higher molecular mass species in the absence of reducing agent, and its ATPase was activated by dithiothreitol.

Investigation of the role of glutamine-471 and glutamine-1114 in the two catalytic sites of P-glycoprotein.

P-glycoprotein, also known as multidrug resistance protein, pumps drugs out of cells using ATP hydrolysis as the energy source. Glutamine-471 and the corresponding glutamine-1114 in the two catalytic sites of P-glycoprotein are conserved in ABC transporters. X-ray structures show that they lie close to the bound nucleotide.