Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, Texas, 75390-8591, USA.
J Biol Chem. 2011 Mar 4;286(9):7308-14. doi: 10.1074/jbc.M110.210880. Epub 2011 Jan 5.
ATP-binding cassette transporters ABCG5 (G5) and ABCG8 (G8) form a heterodimer that transports cholesterol and plant sterols from hepatocytes into bile. Mutations that inactivate G5 or G8 cause hypercholesterolemia and premature atherosclerosis. We showed previously that the two nucleotide-binding domains (NBDs) in the heterodimer are not functionally equivalent; sterol transport is abolished by mutations in the consensus residues of NBD2 but not of NBD1. Here, we examined the structural requirements of NBD1 for sterol transport. Substitutions of the D-loop aspartate and Q-loop glutamine in either NBD did not impair sterol transport. The H-loop histidine of NBD2 (but not NBD1) was required for sterol transport. Exchange of the signature motifs between the NBDs did not interfere with sterol transport, whereas swapping the Walker A, Walker B, and signature motifs together resulted in failure to transport sterols. Selected substitutions within NBD1 altered substrate specificity: transport of plant sterols by the heterodimer was preserved, whereas transport of cholesterol was abolished. In summary, these data indicate that NBD1, although not required for ATP hydrolysis, is essential for normal function of G5G8 in sterol transport. Both the position and structural integrity of NBD2 are essential for sterol transport activity.
三磷酸腺苷结合盒转运蛋白 ABCG5（G5）和 ABCG8（G8）形成异二聚体，将胆固醇和植物甾醇从肝细胞转运到胆汁中。使 G5 或 G8 失活的突变会导致高胆固醇血症和早发性动脉粥样硬化。我们之前曾表明，异二聚体中的两个核苷酸结合域（NBD）在功能上并不等效；NBD2 的保守残基中的突变会使甾醇转运失活，但 NBD1 中的突变不会。在这里，我们研究了 NBD1 对甾醇转运的结构要求。在任一个 NBD 中取代 D 环天冬氨酸和 Q 环谷氨酰胺均不会损害甾醇转运。NBD2 的 H 环组氨酸（而非 NBD1）是甾醇转运所必需的。NBD 之间的特征基序交换不会干扰甾醇转运，而 Walker A、Walker B 和特征基序的一起交换则导致甾醇无法转运。NBD1 内的选定取代改变了底物特异性：异二聚体对植物甾醇的转运得以保留，而胆固醇的转运则被废除。总之，这些数据表明，尽管 NBD1 不参与 ATP 水解，但对于 G5G8 在甾醇转运中的正常功能是必需的。NBD2 的位置和结构完整性对于甾醇转运活性都是必需的。
