Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University , Kyoto 606-8502, Japan.
J Lipid Res. 2012 Jan;53(1):126-36. doi: 10.1194/jlr.M019976. Epub 2011 Oct 25.
ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high-density lipoprotein (HDL) metabolism. Although it is predicted that apolipoprotein A-I (apoA-I) directly binds to ABCA1, the physiological importance of this interaction is still controversial and the conformation required for apoA-I binding is unclear. In this study, the role of the two nucleotide-binding domains (NBD) of ABCA1 in apoA-I binding was determined by inserting a TEV protease recognition sequence in the linker region of ABCA1. Analyses of ATP binding and occlusion to wild-type ABCA1 and various NBD mutants revealed that ATP binds equally to both NBDs and is hydrolyzed at both NBDs. The interaction with apoA-I and the apoA-I-dependent cholesterol efflux required not only ATP binding but also hydrolysis in both NBDs. NBD mutations and cellular ATP depletion decreased the accessibility of antibodies to a hemagglutinin (HA) epitope that was inserted at position 443 in the extracellular domain (ECD), suggesting that the conformation of ECDs is altered by ATP hydrolysis at both NBDs. These results suggest that ATP hydrolysis at both NBDs induces conformational changes in the ECDs, which are associated with apoA-I binding and cholesterol efflux.
三磷酸腺苷结合盒蛋白 A1（ABCA1）在胆固醇稳态和高密度脂蛋白（HDL）代谢中发挥重要作用。虽然预测载脂蛋白 A-I（apoA-I）直接与 ABCA1 结合，但这种相互作用的生理重要性仍存在争议，并且不清楚 apoA-I 结合所需的构象。在这项研究中，通过在 ABCA1 的连接区域插入 TEV 蛋白酶识别序列，确定了 ABCA1 的两个核苷酸结合结构域（NBD）在 apoA-I 结合中的作用。对 ATP 结合和对野生型 ABCA1 和各种 NBD 突变体的封闭分析表明，ATP 平等地结合到两个 NBD 上，并在两个 NBD 上水解。与 apoA-I 的相互作用和 apoA-I 依赖性胆固醇流出不仅需要 ATP 结合，还需要两个 NBD 的水解。NBD 突变和细胞内 ATP 耗竭降低了针对插入在细胞外结构域（ECD）第 443 位的血凝素（HA）表位的抗体的可及性，这表明两个 NBD 上的 ATP 水解改变了 ECD 的构象。这些结果表明，两个 NBD 上的 ATP 水解诱导 ECD 构象发生变化，这与 apoA-I 结合和胆固醇流出有关。
