title: Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein.
abstract: To identify the roles of the two nucleotide-binding folds (NBFs) in the function of human P-glycoprotein, a multidrug transporter, we mutated the key lysine residues to methionines and the cysteine residues to alanines in the Walker A (WA) motifs (the core consensus sequence) in the NBFs. We examined the effects of these mutations on N-ethylmaleimide (NEM) and ATP binding, as well as on the vanadate-induced nucleotide trapping with 8-azido-[alpha-32P]ATP. Mutation of the WA lysine or NEM binding cysteine in either of the NBFs blocked vanadate-induced nucleotide trapping of P-glycoprotein. These results suggest that if one NBF is non-functional, there is no ATP hydrolysis even if the other functional NBF contains a bound nucleotide, further indicating the strong cooperation between the two NBFs of P-glycoprotein. However, we found that the effect of NEM modification at one NBF on ATP binding at the other NBF was not equivalent, suggesting a non-equivalency of the role of the two NBFs in P-glycoprotein function.
authors.affiliation: Laboratory of Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-01, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Takada
authors.forename: K
authors.initials: K
authors.lastname: Yamada
authors.forename: Y
authors.initials: Y
authors.lastname: Taguchi
authors.forename: K
authors.initials: K
authors.lastname: Kino
authors.forename: M
authors.initials: M
authors.lastname: Matsuo
authors.forename: S J
authors.initials: SJ
authors.lastname: Tucker
authors.forename: T
authors.initials: T
authors.lastname: Komano
authors.forename: T
authors.initials: T
authors.lastname: Amachi
authors.forename: K
authors.initials: K
authors.lastname: Ueda
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D005033:Ethylmaleimide / Q000737:chemistry; D006801:Humans; D011485:Protein Binding; D017510:Protein Folding; D014638:Vanadates / Q000037:antagonists & inhibitors / Q000737:chemistry
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D014638:Vanadates; D000255:Adenosine Triphosphate; D003545:Cysteine; D005033:Ethylmaleimide