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 ex pression 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 t echniques were used to probe the nucleotide binding domains (NBD) of P -glycoprotein. The transporter was labeled at two conserved cysteine r esidues, one within each NBD, using the thiol-reactive fluor 2-(4'-mal eimidylanilino)-naphthalene-6-sulfonic acid (MIANS), and collisional q uenching 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 hig hly effective quencher, whereas Cs+ was not, demonstrating the presenc e of a positive charge in the region close to the ATP binding site. Th e fluorescent nucleotide derivative 2'(3')-O-(2,4,6-trinitrophenyl)-AT P (TNP-ATP) was hydrolysed slowly by P-glycoprotein, with a V-max simi lar to 20-fold lower than that for unmodified ATP, and a K-M of 81 mu M. TNP-ATP and TNP-ADP inhibited P-glycoprotein ATPase activity, indic ating that they interact with the NBD, whereas TNP-AMP was a very poor inhibitor. When TNP-nucleotides bound to P-glycoprotein, their fluore scence intensity was enhanced in a concentration-dependent manner. Bot h TNP-ATP and TNP-ADP bound to P-glycoprotein with substantially highe r affinity than ATP, with K-d values of 43 and 36 mu M, 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 clos e to each other within the catalytic site of P-glycoprotein.