Identification of the Cyclosporin-Binding Site in P-Glycoprotein

Abstract

The binding site of cyclosporin A to P-glycoprotein was characterized by using a multidrug-resistant Chinese hamster ovary cell line. P-glycoprotein photolabeled with diazirine-cyclosporin A analogue was purified by a two-step process involving continuous elution electrophoresis followed by wheat germ agglutinin-agarose precipitation. The cyclosporin A covalently bound to P-glycoprotein and to subsequent proteolytic fragments was detected by Western blot analysis using a monoclonal antibody against cyclosporin A. Proteolytic digestion of purified P-glycoprotein by V8 generated a major fragment of 15 kDa photolabeled by cyclosporin A, while proteolysis of P-glycoprotein photolabeled by [125I]-iodoaryl azidoprazosin generated a major fragment of 7 kDa. Limited proteolysis of cyclosporin A-photolabeled P-glycoprotein with trypsin indicated that the major binding site for cyclosporin A was in the C-terminal half of the protein. This cyclosporin A binding site was further characterized with chemical agents (N-chlorosuccinimide, cyanogen bromide, and 2-nitro-5-thiocyanobenzoate). These three chemical agents established a proteolytic profile of P-glycoprotein for fragments photolabeled with cyclosporin A and for fragments that contained the C494 and C219 epitopes. The smallest fragments generated by these chemical agents include the transmembrane domains (TMs) 10, 11, and 12 of P-glycoprotein. When the fragments generated by these chemical agents are aligned, the region that binds cyclosporin A is reduced to the 953-1007 residues. These combined results suggest that the major binding site of cyclosporin A occurs between the end of TM 11 and the end of TM 12.

Large Scale Purification of Detergent-soluble P-glycoprotein fromPichia pastoris Cells and Characterization of Nucleotide Binding Properties of Wild-type, Walker A, and Walker B Mutant Proteins

“…Subsequent reports (12)(13)(14)(15)(16) have supported and extended this proposed mechanism. Further work (17)(18)(19)(20) on the structure and location of the drug-binding sites has shown that certain transmembrane ␣-helices are involved in forming these sites, and work (21) with the closely related LmrA protein showed that transport is indeed from the inner lipid leaflet to the outer surface as proposed in Ref. 22.…”

Correlation between Steady-state ATP Hydrolysis and Vanadate-induced ADP Trapping in Human P-glycoprotein

“…Unfortunately, values of K app for these drugs are not able to be quantitated because of the high concentrations (0.4 M) of Pgp required to assay for activity in either the cycling or P i assay. However, data describing cyclosporin A as an inhibitor report low concentrations (74 -400 nM) for maximal effect (6,33,40,41) so the saturation of these compounds is likely.…”

The Size of a Single Residue of the Sulfonylurea Receptor Dictates the Effectiveness of KATP Channel Openers

“…The broad specificity of SUR2 for openers; the role of TM helix 17, which has been implicated in binding of substrates in other polyspecific ABC transporters such as MRP1 (Daoud et al, 2001;Ito et al, 2001a;Zhang et al, 2001;Mao et al, 2002;Ren et al, 2002;Karwatsky et al, 2003), MRP3 (Oleschuk et al, 2003;Zhang et al, 2003), and multidrug resistance protein 1 (Loo and Clarke 1997Demeule et al, 1998;Dey et al, 1999); and the link between openers and ATPase activity of SUR2A (Bienengraeber et al, 2000) led to the hypothesis that openers could act as pseudosubstrates of SUR by occupying a substrate-binding pocket structurally conserved in SUR and multidrug resistance proteins.…”