Sequence Requirements of the ATP-Binding Site within the C-Terminal Nucleotide-Binding Domain of Mouse P-Glycoprotein: Structure-Activity Relationships for Flavonoid Binding
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- 作者：Heidi de Wet ; David B. McIntosh ; Gwenaë ; lle Conseil ; Hé ; lè ; ne Baubichon-Cortay ; Tino Krell ; Jean-Michel Jault ; Jean-Baptiste Daskiewicz ; Denis Barron ; and Attilio Di Pietro
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：August 28, 2001
- 年：2001
- 卷：40
- 期：34
- 页码：10382 - 10391
- 全文大小：136K
- 年卷期：v.40,no.34(August 28, 2001)
- ISSN：1520-4995
文摘
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 proteinsof different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N3-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N3-ATP). The two proteins, Thr1044-Thr1224(NBD2short) and Lys1025-Ser1276 (NBD2long), both incorporated the four consensus sequences of ABC(ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, whilediffering in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins wascharacterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K0.5(8N3-ATP) = 36-37 
M and K0.5(TNP-8N3-ATP) = 0.8-2.6 M and was maximal at acidic pH.Photolabeling was strongly inhibited by TNP-ATP (KD = 0.1-5 M) and ATP (KD = 0.5-2.7 mM).Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interactinghydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and DiPietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from differentclasses were investigated for structure-activity relationships toward binding to the ATP site, monitoredby protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appearedimportant, whereas opening of ring C abolished the binding in all but one case. It can be concluded thatthe benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosylsite. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficientto constitute the ATP site.
M and K0.5(TNP-8N3-ATP) = 0.8-2.6 M and was maximal at acidic pH.Photolabeling was strongly inhibited by TNP-ATP (KD = 0.1-5 M) and ATP (KD = 0.5-2.7 mM).Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interactinghydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and DiPietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from differentclasses were investigated for structure-activity relationships toward binding to the ATP site, monitoredby protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appearedimportant, whereas opening of ring C abolished the binding in all but one case. It can be concluded thatthe benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosylsite. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficientto constitute the ATP site.