Determining the Dimensions of the Drug-binding Domain of Human P-glycoprotein Using Thiol Cross-linking Compounds as Molecular Rulers

• Published in: The Journal of biological chemistry 2001-10, Vol.276 (40), p.36877-36880
• Main Authors: Loo, Tip W., Clarke, David M.
• Format: Article
• Language: English
• Subjects: ATP Binding Cassette Transporter, Subfamily B, Member 1 - chemistry; ATP Binding Cassette Transporter, Subfamily B, Member 1 - metabolism; Cells, Cultured; Cross-Linking Reagents - metabolism; Humans; Protein Structure, Tertiary; Sulfhydryl Compounds - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.C100467200

The human multidrug resistance P-glycoprotein (P-gp) interacts with a broad range of compounds with diverse structures and sizes. There is considerable 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-binding site by using thiol-specific methanethiosulfonate (MTS) cross-linkers containing spacer arms of 2 to 17 atoms. The majority of these cross-linkers 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 introduced into transmembrane segments 4–6 and 10–12 were analyzed after reaction with 0.2 mm MTS cross-linker at 4 °C. The cross-linked product migrated with lower mobility than native P-gp in SDS gels. 13 P-gp mutants were cross-linked by MTS cross-linkers with spacer arms of 9–25 Å. Vinblastine and cyclosporin A inhibited cross-linking. The emerging picture from these results and other studies is that the drug-binding domain is large enough to accommodate compounds of different sizes and that the drug-binding domain is “funnel” shaped, narrow at the cytoplasmic side, at least 9–25 Å in the middle, and wider still at the extracellular surface.