Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Doctoral School of Molecular Cell and Immune Biology, University of Debrecen, Debrecen, Hungary.
Elife. 2023 Feb 10;12:e83976. doi: 10.7554/eLife.83976.
ABCG2 is an exporter-type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here, we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.
ABCG2 是一种外排型 ABC 蛋白，能够将许多化学上无关的外源性和内源性物质从细胞中排出。当在肿瘤细胞或肿瘤干细胞中表达时，ABCG2 赋予多药耐药性，导致化疗失败。协调底物转运和 ATP 水解的分子细节仍然难以捉摸。在这里，我们提出了同时研究 ABCG2 在细胞中结合底物和核苷酸的方法。我们使用构象敏感抗体 5D3 表明，ABCG2 从内向（IF）到外向（OF）构象的转变是由核苷酸结合诱导的。IF-OF 转变由底物促进，由抑制剂 Ko143 阻碍。对 5D3 和底物与 ABCG2 结合的直接测量表明，药物结合位点的高到低亲和力转变与从 IF 到 OF 构象的转变一致。低底物结合在水解后状态下持续存在，这支持 ATP 水解产物的解离对于重置 ABCG2 的高底物亲和力 IF 构象是必需的。
