Fluorescence studies on the nucleotide binding domains of the P-glycoprotein multidrug transporter.
One of the major causes of multidrug resistance in human cancers is expression of the P-glycoprotein multidrug transporter, which acts as an efflux pump for a diverse range of natural products, chemotherapeutic drugs, and hydrophobic peptides. In the present study, fluorescence techniques were used to probe the nucleotide binding domains (NBD) of P-glycoprotein. The transporter was labeled at two conserved cysteine residues, one within each NBD, using the thiol-reactive fluor 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS), and collisional quenching was used to assess solvent accessibility of the bound probe. Acrylamide was a poor quencher, which suggests that MIANS is buried in a relatively inaccessible region of the protein. Iodide ion was a highly effective quencher, whereas Cs+ was not, demonstrating the presence of a positive charge in the region close to the ATP binding site. The fluorescent nucleotide derivative 2'(3')-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) was hydrolysed slowly by P-glycoprotein, with a V(max) approximately 20-fold lower than that for unmodified ATP, and a K(M) of 81 microM. TNP-ATP and TNP-ADP inhibited P-glycoprotein ATPase activity, indicating that they interact with the NBD, whereas TNP-AMP was a very poor inhibitor. When TNP-nucleotides bound to P-glycoprotein, their fluorescence intensity was enhanced in a concentration-dependent manner. Both TNP-ATP and TNP-ADP bound to P-glycoprotein with substantially higher affinity than ATP, with K(d) values of 43 and 36 microM, respectively. Addition of ATP led to only partial displacement of TNP-ATP. Resonance energy transfer was observed between cysteine-bound MIANS and TNP-ATP/ADP, which indicated that the two fluorescent groups are located close to each other within the catalytic site of P-glycoprotein.
Liu
R
R
Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry and Biochemistry, University of Guelph, Ontario, Canada.
Sharom
F J
FJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism; D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001665:Binding Sites; D002586:Cesium / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D007454:Iodides / Q000494:pharmacology; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000178:Acrylamides; D000817:Anilino Naphthalenesulfonates; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007454:Iodides; D009711:Nucleotides; D013439:Sulfhydryl Reagents; D002586:Cesium; D020106:Acrylamide; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases