School of Natural Sciences, University of California, Merced, 4225 N. Hospital Road, Atwater, CA, USA.
Department of Cell Physiology and Molecular Biophysics, and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX 79423-6551, USA.
Biochim Biophys Acta Biomembr. 2018 Apr;1860(4):854-867. doi: 10.1016/j.bbamem.2017.08.005. Epub 2017 Aug 8.
The ATP-binding cassette (ABC) superfamily includes regulatory and transport proteins. Most human ABC exporters pump substrates out of cells using energy from ATP hydrolysis. Although major advances have been made toward understanding the molecular mechanism of ABC exporters, there are still many issues unresolved. During the last few years, luminescence resonance energy transfer has been used to detect conformational changes in real time, with atomic resolution, in isolated ABC nucleotide binding domains (NBDs) and full-length ABC exporters. NBDs are particularly interesting because they provide the power stroke for substrate transport. Luminescence resonance energy transfer (LRET) is a spectroscopic technique that can provide dynamic information with atomic-resolution of protein conformational changes under physiological conditions. Using LRET, it has been shown that NBD dimerization, a critical step in ABC proteins catalytic cycle, requires binding of ATP to two nucleotide binding sites. However, hydrolysis at just one of the sites can drive dissociation of the NBD dimer. It was also found that the NBDs of the bacterial ABC exporter MsbA reconstituted in a lipid bilayer membrane and studied at 37°C never separate as much as suggested by crystal structures. This observation stresses the importance of performing structural/functional studies of ABC exporters under physiologic conditions. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.
ATP 结合盒（ABC）超家族包括调节蛋白和转运蛋白。大多数人类 ABC 外排泵利用 ATP 水解产生的能量将底物泵出细胞。尽管人们在理解 ABC 外排泵的分子机制方面取得了重大进展，但仍有许多问题尚未解决。在过去的几年中，荧光共振能量转移已被用于实时、原子分辨率地检测分离的 ABC 核苷酸结合域（NBD）和全长 ABC 外排泵中的构象变化。NBD 特别有趣，因为它们为底物转运提供动力冲程。荧光共振能量转移（LRET）是一种光谱技术，可以在生理条件下提供蛋白质构象变化的原子分辨率的动态信息。使用 LRET，已经表明 NBD 二聚化是 ABC 蛋白催化循环中的关键步骤，需要 ATP 结合到两个核苷酸结合位点。然而，仅在一个位点的水解就可以驱动 NBD 二聚体的解离。还发现，在 37°C 下在脂质双层膜中重建的细菌 ABC 外排泵 MsbA 的 NBD 从未像晶体结构所暗示的那样分离得那么多。这一观察结果强调了在生理条件下对 ABC 外排泵进行结构/功能研究的重要性。本文是由 Ute Hellmich、Rupak Doshi 和 Benjamin McIlwain 编辑的特刊“超越膜蛋白的结构-功能范围”的一部分。
