School of Life Sciences, GIST, 123 Cheomdan-gwagiro, Buk-gu, Gwangju, 61005, Republic of Korea.
Department of Life Sciences, POSTECH, 77 Cheongam-Ro, Nam-gu, Pohang, 37673, Republic of Korea.
Commun Biol. 2023 Sep 21;6(1):960. doi: 10.1038/s42003-023-05339-3.
Human ATP-binding cassette transporter subfamily B6 (ABCB6) is a mitochondrial ATP-driven pump that translocates porphyrins from the cytoplasm into mitochondria for heme biosynthesis. Within the transport pathway, a conserved aromatic residue W546 located in each monomer plays a pivotal role in stabilizing the occluded conformation via π-stacking interactions. Herein, we employed cryo-electron microscopy to investigate the structural consequences of a single W546A mutation in ABCB6, both in detergent micelles and nanodiscs. The results demonstrate that the W546A mutation alters the conformational dynamics of detergent-purified ABCB6, leading to entrapment of the transporter in an outward-facing transient state. However, in the nanodisc system, we observed a direct interaction between the transporter and a phospholipid molecule that compensates for the absence of the W546 residue, thereby facilitating the normal conformational transition of the transporter toward the occluded state following ATP hydrolysis. The findings also reveal that adoption of the outward-facing conformation causes charge repulsion between ABCB6 and the bound substrate, and rearrangement of key interacting residues at the substrate-binding site. Consequently, the affinity for the substrate is significantly reduced, facilitating its release from the transporter.
人源 ATP 结合盒转运蛋白亚家族 B6（ABCB6）是一种线粒体 ATP 驱动泵，可将卟啉从细胞质转运到线粒体中用于血红素生物合成。在转运途径中，每个单体中保守的芳香族残基 W546 通过π堆积相互作用在稳定被阻断构象方面发挥关键作用。在此，我们使用冷冻电子显微镜研究了 ABCB6 中单个 W546A 突变的结构后果，包括在去污剂胶束和纳米圆盘中的结构后果。结果表明，W546A 突变改变了去污剂纯化的 ABCB6 的构象动力学，导致转运蛋白被捕获在外向瞬变状态。然而，在纳米圆盘系统中，我们观察到转运蛋白与磷脂分子之间的直接相互作用，该相互作用补偿了 W546 残基的缺失，从而促进了转运蛋白在 ATP 水解后向被阻断状态的正常构象转变。研究结果还表明，采用外向构象会导致 ABCB6 与结合底物之间的电荷排斥，并导致底物结合位点上关键相互作用残基的重排。因此，对底物的亲和力显著降低，从而促进了底物从转运蛋白中的释放。
