Molecular Microscopy Research Group, Zernike Institute for Advanced Material, University of Groningen, 9747 AG Groningen, the Netherlands; Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg-Martinsried, Germany; Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Molecular Bacteriology, KU Leuven, 3000 Leuven, Belgium.
Institute of Biochemistry, Biocenter, Goethe University Frankfurt, 60438 Frankfurt a.M., Germany.
Cell Rep. 2019 Jul 16;28(3):723-734.e6. doi: 10.1016/j.celrep.2019.06.052.
The twin-ATPase ABCE1 has a vital function in mRNA translation by recycling terminated or stalled ribosomes. As for other functionally distinct ATP-binding cassette (ABC) proteins, the mechanochemical coupling of ATP hydrolysis to conformational changes remains elusive. Here, we use an integrated biophysical approach allowing direct observation of conformational dynamics and ribosome association of ABCE1 at the single-molecule level. Our results from FRET experiments show that the current static two-state model of ABC proteins has to be expanded because the two ATP sites of ABCE1 are in dynamic equilibrium across three distinct conformational states: open, intermediate, and closed. The interaction of ABCE1 with ribosomes influences the conformational dynamics of both ATP sites asymmetrically and creates a complex network of conformational states. Our findings suggest a paradigm shift to redefine the understanding of the mechanochemical coupling in ABC proteins: from structure-based deterministic models to dynamic-based systems.
双 ATP 酶 ABCE1 在回收终止或停滞的核糖体方面具有重要的 mRNA 翻译功能。对于其他具有不同功能的 ATP 结合盒（ABC）蛋白，ATP 水解与构象变化的机械化学偶联仍然难以捉摸。在这里，我们使用一种集成的生物物理方法，允许在单分子水平上直接观察 ABCE1 的构象动力学和核糖体缔合。我们的 FRET 实验结果表明，当前的 ABC 蛋白静态两态模型必须扩展，因为 ABCE1 的两个 ATP 结合位点在三个不同的构象状态之间处于动态平衡：开放、中间和关闭。ABCE1 与核糖体的相互作用不对称地影响两个 ATP 结合位点的构象动力学，并创建一个复杂的构象状态网络。我们的发现表明，需要对 ABC 蛋白的机械化学偶联的理解进行范式转变：从基于结构的确定性模型到基于动态的系统。
