Determining the dimensions of the drug-binding domain of human P-glycoprotein using thiol cross-linking compounds as molecular rulers

The human multidrug resistance P-glycoprotein (P-gp) interacts with a broad range of compounds with diverse structures and sizes. There is considerabl e evidence indicating that residues in transmembrane segments 4-6 and 10-12 form the drug-binding site. We attempted to measure the size of the drug-b inding site by using thiol-specific methanethiosulfonate (MTS) cross-linker s containing spacer arms of 2 to 17 atoms. The majority of these cross-link ers were also substrates of P-gp, because they stimulated ATPase activity ( 2.5- to 10.1-fold). 36 P-gp mutants with pairs of cysteine residues introdu ced into transmembrane segments 4-6 and 10-12 were analyzed after reaction with 0.2 mm MTS cross-linker at 4 degreesC. The cross-linked product migrat ed with lower mobility than native P-gp in SDS gels. 13 P-gp mutants were c ross-linked by MTS cross-linkers with spacer arms of 9-25 Angstrom Vinblast ine and cyclosporin A inhibited crosslinking. The emerging picture from the se results and other studies is that the drug-binding domain is large enoug h to accommodate compounds of different sizes and that the drug-binding dom ain is "funnel" shaped, narrow at the cytoplasmic side, at least 9-25 Angst rom in the middle, and wider still at the extracellular surface.