Abstract
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 degrees 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 A. 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 A in the middle, and wider still at the extracellular surface.
Highlights
Determining the Dimensions of the Drug-binding Domain of Human P-glycoprotein Using Thiol Cross-linking Compounds as Molecular Rulers*
Its 1280 amino acids are organized in two repeating units of 610 amino acids that are joined by a linker region of about 60 amino acids [9]
The minimal functional unit in P-gp is a monomer [12]. Both nucleotidebinding sites are required for function, because P-gp is inactive when ATP hydrolysis at either site in blocked by mutation or chemical modification [13,14,15,16,17,18]
Summary
P-gp, P-glycoprotein; MTS, methanethiosulfonate; TM, transmembrane; PAGE, polyacrylamide gel electrophoresis; HEK, human embryonic kidney; M2M, 1,2-ethanediyl bismethanethiosulfonate; M3M, 1,3-propanediyl bismethanethiosulfonate; M4M, 1,4-butanediyl bismethanethiosulfonate; M5M, 1,5-pentanediyl bismethanethiosulfonate; M6M, 1,6-hexanediyl bismethanethiosulfonate; M8M, 3,6-dioxaoctane-1,8-diyl bismethanethiosulfonate; M11M, 3,6,9-trioxaundecane-1,11-diyl bismethanethiosulfonate; M14M, 3,6,9,12-tetraoxatetradecane-1,14-diyl bismethanethiosulfonate; ATP to pump out of the cell a wide variety of structurally diverse compounds (recently reviewed in Ref. 1). Photolabeling and mutational studies indicate that the drugbinding domain is within the TM domains [21,22,23,24,25,26,27,28,29,30,31] This is supported by the finding that a deletion mutant lacking both nucleotide-binding domains can still bind drug substrate [32]. We used a series of thiol-specific cross-linkers with spacer arms of various lengths to measure distances between these residues
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