adenosine-5--o-(3-thiotriphosphate) and 2-(4--maleimidylanilino)naphthalene-6-sulfonic-acid

P-glycoprotein (ABCB1), a member of the ABC superfamily, functions as an ATP-driven multidrug efflux pump. The catalytic cycle of ABC proteins is believed to involve formation of a sandwich dimer in which two ATP molecules are bound at the interface of the nucleotide binding domains (NBDs). However, such dimers have only been observed in isolated NBD subunits and catalytically arrested mutants, and it is still not understood how ATP hydrolysis is coordinated between the two NBDs. We report for the first time the characterization of an asymmetric state of catalytically active native P-glycoprotein with two bound molecules of adenosine 5'-(gamma-thio)triphosphate (ATPgammaS), one of low affinity (K(d) 0.74 mm), and one "occluded" nucleotide of 120-fold higher affinity (K(d) 6 microm). ATPgammaS also interacts with P-glycoprotein with high affinity as assessed by inhibition of ATP hydrolysis and protection from covalent labeling of a Walker A Cys residue, whereas other non-hydrolyzable ATP analogues do not. Binding of ATPgammaS (but not ATP) causes Trp residue heterogeneity, as indicated by collisional quenching, suggesting that it may induce conformational asymmetry. Asymmetric ATPgammaS-bound P-glycoprotein does not display reduced binding affinity for drugs, implying that transport is not driven by ATP binding and likely takes place at a later stage of the catalytic cycle. We propose that this asymmetric state with two bound nucleotides represents the next intermediate on the path toward ATP hydrolysis after nucleotide binding, and an alternating sites mode of action is achieved by simultaneous switching of the two active sites between high and low affinity states.

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Affinity Labels; Anilino Naphthalenesulfonates; Animals; Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Binding Sites; Catalysis; Cricetinae; Cricetulus; Daunorubicin; Humans; Mice; Nucleotides; Protein Binding; Protein Structure, Tertiary; Tubulin Modulators; Vanadates; Vinblastine

P-glycoprotein (P-gp) confers multiple drug resistance on cancer cells by acting as a plasma membrane localized ATP-dependent drug efflux pump. Currently, there is little information on the nature of the communication between the energy-providing nucleotide binding domains (NBDs) and the drug binding sites of P-gp to generate transport of substrate. Many substrates and modulators cause alterations in ATP hydrolysis, but what effect do the various stages of the catalytic cycle have on drug interaction with P-gp? Vanadate trapping of Mg.ADP caused a reversible decrease in the binding capacity of the transported substrate [(3)H]-vinblastine and the nontransported modulator [(3)H]XR9576 to P-gp in CH(r)B30 cell membranes. The non-hydrolyzable nucleotide analogue ATP-gamma-S also caused a reduction in the binding capacity of [(3)H]-vinblastine but not for the modulator [(3)H]XR9576. This indicates that signaling to the NBDs following binding of a nontransported modulator is different to that transmitted upon interaction of a transported substrate. Second, it appears that the binding of nucleotide, rather than its hydrolysis, causes the initial conformational shift in the drug-binding site during a transport cycle.

Topics: 4-Chloro-7-nitrobenzofurazan; Adenosine Triphosphatases; Adenosine Triphosphate; Anilino Naphthalenesulfonates; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Binding Sites; CHO Cells; Cricetinae; Enzyme Activation; Enzyme Inhibitors; Ethylmaleimide; Hydrolysis; Substrate Specificity; Vanadates