期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 277, 期 46, 页码 44332-44338出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M208433200
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资金
- NCI NIH HHS [R01 CA80900] Funding Source: Medline
The human multidrug resistance P-glycoprotein (P-gp) pumps a wide variety of structurally diverse compounds out of the cell. It is an ATP-binding cassette transporter with two nucleotide-binding domains and twos transmembrane (TM) domains. One class of compounds transported by P-gp is the rhodamine dyes. A P-gp deletion mutant (residues 1-379 plus 681-1025) With only the TM domains retained the ability to bind rhodamine. Therefore, to identify the residues involved in rhodamine binding, 252 mutants containing a cysteine in the predicted TM segments were generated and reacted with a thiol-reactive analog of rhodamine, methanethiosulfonate (MTS)-rhodamine. The activities of 28 mutants (in TMs 2-12) were inhibited by at least 50% after reaction with MTS-rhodamine. The activities of five mutants, 1340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12), however, were significantly protected from inhibition by MTS-rhodamine by pretreatment with rhodamine B, indicating that residues in TMs 6, 9, and 12 contribute to the binding of rhodamine dyes. These results, together with those from previous labeling studies with other thiol-reactive compounds, dibromobimane, MTS-verapamil, and MTS-cross-linker substrates, indicate that common residues are involved in the binding of structurally different drug substrates and that P-gp has a common drug-binding site. The results support the substrate-induced fit hypothesis for drug binding.
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