Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein.
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.
Takada
Y
Y
Laboratory of Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-01, Japan.
Yamada
K
K
Taguchi
Y
Y
Kino
K
K
Matsuo
M
M
Tucker
S J
SJ
Komano
T
T
Amachi
T
T
Ueda
K
K
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
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D014638:Vanadates; D000255:Adenosine Triphosphate; D003545:Cysteine; D005033:Ethylmaleimide