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

Sequence requirements of the ATP-binding site within the C-terminal nucleot ide-binding domain (NBD2) of mouse P-glycoprotein were investigated by usin g two recombinantly expressed soluble proteins of different lengths and pho toactive 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)-Se r(1276) (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, 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 muM and K-0.5(TNP-8N(3)-ATP) = 0.8-2.6 muM and was ma ximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K-D = 0.1-5 muM) and ATP (K-D = 0.5-2.7 mM). Since flavonoids display bifunctiona l interactions at the ATP-binding site and a vicinal steroid-interacting hy drophobic 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 investig ated for structure-activity relationships toward binding to the ATP site, m onitored by protection against photolabeling. The 3-OH and aromaticity of c onjugated rings A and C appeared important, whereas opening of ring C aboli shed the binding in all but one case. It can be concluded that the benzopyr one 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, an d small segments on both sides are sufficient to constitute the ATP site.