title: Characterization of an asymmetric occluded state of P-glycoprotein with two bound nucleotides: implications for catalysis.
abstract: 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.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Alena
authors.initials: A
authors.lastname: Siarheyeva
authors.forename: Ronghua
authors.initials: R
authors.lastname: Liu
authors.forename: Frances J
authors.initials: FJ
authors.lastname: Sharom
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000345:Affinity Labels / Q000737:chemistry / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D006224:Cricetinae; D003412:Cricetulus; D003630:Daunorubicin / Q000378:metabolism; D006801:Humans; D051379:Mice; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050257:Tubulin Modulators / Q000378:metabolism; D014638:Vanadates / Q000378:metabolism; D014747:Vinblastine / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D000817:Anilino Naphthalenesulfonates; D000903:Antibiotics, Antineoplastic; D009711:Nucleotides; D050257:Tubulin Modulators; D000266:Adenylyl Imidodiphosphate; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; C022571:adenosine 5'-O-(3-thiotriphosphate); D014638:Vanadates; D014747:Vinblastine; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D000255:Adenosine Triphosphate; D003630:Daunorubicin