Stoichiometry and affinity of nucleotide binding to P-glycoprotein during the catalytic cycle.
Drug transport mediated by P-glycoprotein (Pgp) is driven by hydrolysis of ATP at the two cytosolic nucleotide binding domains. However, little is currently known concerning the stoichiometry of nucleotide binding and how both stoichiometry and binding affinity change during the catalytic cycle of the transporter. To address this issue, we used fluorescence techniques to measure both the number of nucleotides bound to P-glycoprotein during various stages of the catalytic cycle and the affinity of nucleotide binding. Results showed that resting state P-glycoprotein bound two molecules of the fluorescent nucleotide derivative, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), whereas the vanadate-trapped transition state bound only one nucleotide molecule. Both resting and transition state P-glycoprotein showed similar affinity for TNP-ATP/TNP-ADP and unlabeled ATP/ADP. Following binding of various drugs, resting state P-glycoprotein displayed a higher affinity for nucleotides, up to 4-fold depending on the compound used. In contrast, the transition state showed substantially lower (up to 3-fold) nucleotide binding affinity when the drug binding site(s) is/are occupied. These results indicate that both nucleotide binding domains of P-glycoprotein are likely to be occupied with either ATP (or ADP) in the resting state and the transition state in the absence of transport substrates. Drugs alter the binding affinity to favor association of ATP with P-glycoprotein at the start of the catalytic cycle and release of ADP from the transition state following nucleotide hydrolysis.
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Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Russell
Paula L
PL
Sharom
Frances J
FJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002384:Catalysis; D006224:Cricetinae; D005456:Fluorescent Dyes / Q000378:metabolism; D006868:Hydrolysis; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014301:Trinitrobenzenes / Q000378:metabolism; D014638:Vanadates / Q000737:chemistry
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D014301:Trinitrobenzenes; C029222:sym-trinitrobenzene; D014638:Vanadates; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000244:Adenosine Diphosphate; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D000255:Adenosine Triphosphate