de Wet H, McIntosh D B, Conseil G, Baubichon-Cortay H, Krell T, Jault J M, Daskiewicz J B, Barron D, Di Pietro A
Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, Cape Town, South Africa.
Biochemistry. 2001 Aug 28;40(34):10382-91. doi: 10.1021/bi010657c.
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.
通过使用两种不同长度的重组表达可溶性蛋白以及光活性ATP类似物8-叠氮三磷酸腺苷（8N(3)-ATP）和2',3',4'-O-(2,4,6-三硝基苯基)-8-叠氮三磷酸腺苷（TNP-8N(3)-ATP），研究了小鼠P-糖蛋白C端核苷酸结合结构域（NBD2）内ATP结合位点的序列要求。这两种蛋白，即苏氨酸（1044）-苏氨酸（1224）（NBD2(短)）和赖氨酸（1025）-丝氨酸（1276）（NBD2(长)），都包含ABC（ATP结合盒）转运蛋白的四个共有序列、沃克A和B基序、Q环以及ABC特征序列，只是在N端和C端延伸部分有所不同。两种蛋白的放射性光标记表现出对核苷酸浓度的双曲线依赖性以及与K(0.5)(8N(3)-ATP)=36 - 37微摩尔和K(0.5)(TNP-8N(3)-ATP)=0.8 - 2.6微摩尔的高亲和力结合，并且在酸性pH下达到最大值。光标记受到TNP-ATP（K(D)=0.1 - 5微摩尔）和ATP（K(D)=0.5 - 2.7毫摩尔）的强烈抑制。由于黄酮类化合物在ATP结合位点表现出双功能相互作用以及一个相邻的类固醇相互作用疏水序列[孔塞尔，G.，鲍比雄 - 科尔泰，H.，达扬，G.，若尔，J.-M.，巴伦，D.，和迪彼得罗，A.（1998年）美国国家科学院院刊95，9831 - 9836]，研究了一系列30种不同类别的黄酮类化合物与ATP位点结合的构效关系，通过防止光标记来监测。共轭环A和C的3 - OH和芳香性显得很重要，而除了一种情况外，环C的打开都消除了结合。可以得出结论，黄酮类化合物的苯并吡喃部分结合在腺苷位点，苯环B结合在核糖位点。沃克A和B基序、中间序列以及两侧的小片段足以构成ATP位点。
