Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding.

De Wet, H; McIntosh, D; Conseil, G; Baubichon-Cortay, H; Krell, T; Jault, J; Daskiewicz, J; Barron, D; Di Pietro, A

Journal article

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding.

Abstract:: Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.

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APA Style

De Wet, H., McIntosh, D., Conseil, G., Baubichon-Cortay, H., Krell, T., Jault, J., Daskiewicz, J., Barron, D., & Di Pietro, A. (2001). Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding. Biochemistry, 40(34), 10382–10391.

MLA Style

De Wet, H, et al. “Sequence Requirements of the ATP-Binding Site within the C-Terminal Nucleotide-Binding Domain of Mouse P-Glycoprotein: Structure-Activity Relationships for Flavonoid Binding.” Biochemistry, vol. 40, no. 34, 2001, pp. 10382–91.

Chicago Style

De Wet, H, D McIntosh, G Conseil, et al. 2001. “Sequence Requirements of the ATP-Binding Site within the C-Terminal Nucleotide-Binding Domain of Mouse P-Glycoprotein: Structure-Activity Relationships for Flavonoid Binding.” Biochemistry 40 (34): 10382–91.
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