SITE-DIRECTED FLUORESCENCE LABELING OF P-GLYCOPROTEIN ON CYSTEINE RESIDUES IN THE NUCLEOTIDE-BINDING DOMAINS

P-Glycoprotein is a member of the ABC superfamily of membrane transpor ters, and functions as an ATP-driven active efflux pump for natural pr oducts and chemotherapeutic drugs. Overexpression of P-glycoprotein is a major cause of multidrug resistance in human cancers. Sulfhydryl mo dification agents are known to inactivate both P-glycoprotein ATPase a ctivity and transport function. In the present study, P-glycoprotein p urified from CH(R)B30 cells was covalently labeled at two conserved Cy s residues, one within each of the nucleotide binding domains, using 2 -(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS). MIANS modifi cation inactivated P-glycoprotein ATPase function, in a concentration- dependent fashion. Increasing concentrations of ATP blocked MIANS labe ling with an IC50 of 0.37 mM (similar to the K-M for ATP hydrolysis), which suggests that the label is located close to the site of ATP bind ing within the nucleotide binding domain. A blue shift in the fluoresc ence spectrum of MIANS bound to P-glycoprotein indicated that the labe led Cys residues are situated in a nonpolar environment. MIANS-labeled P-glycoprotein was still able to bind ATP, as demonstrated by quenchi ng of the fluorescence, with a K-d of 0.46 mM. Addition of a variety o f drugs and chemosensitizers to MIANS-labeled P-glycoprotein led to su bstantial quenching of the probe fluorescence within the nucleotide bi nding domains. Dissociation constants for drug binding measured by flu orescence quenching were in the range of 0.77 mu M for vinblastine to 158 mu M for colchicine. Quenching by ATP and drugs was independent an d additive, suggesting that each produces a defined change in the prot ein. The rate of MIANS labeling of Pgp was reduced in the presence of drugs and chemosensitizers, implying that a long-range conformational change arises from drug binding which alters the accessibility of the nucleotide binding domains to MIANS. These results suggest that there is conformational communication between the drug binding site(s) of P- glycoprotein and the ATPase catalytic sites within the nucleotide bind ing domains.