1] Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands. [2] Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands.
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands.
Nat Struct Mol Biol. 2015 Jan;22(1):57-64. doi: 10.1038/nsmb.2929. Epub 2014 Dec 8.
The conformational dynamics in ABC transporters is largely elusive. The ABC importer GlnPQ from Lactococcus lactis has different covalently linked substrate-binding domains (SBDs), thus making it an excellent model system to elucidate the dynamics and role of the SBDs in transport. We demonstrate by single-molecule spectroscopy that the two SBDs intrinsically transit from open to closed ligand-free conformation, and the proteins capture their amino acid ligands via an induced-fit mechanism. High-affinity ligands elicit transitions without changing the closed-state lifetime, whereas low-affinity ligands dramatically shorten it. We show that SBDs in the closed state compete for docking onto the translocator, but remarkably the effect is strongest without ligand. We find that the rate-determining steps depend on the SBD and the amino acid transported. We conclude that the lifetime of the closed conformation controls both SBD docking to the translocator and substrate release.
ABC 转运蛋白的构象动力学在很大程度上难以捉摸。乳球菌 lactis 的 ABC 进口 GlnPQ 具有不同的共价连接的底物结合域 (SBD)，因此使其成为阐明 SBD 在运输中的动力学和作用的极佳模型系统。我们通过单分子光谱证明，两个 SBD 本质上从开放的无配体构象转变为闭合的无配体构象，并且蛋白质通过诱导契合机制捕获其氨基酸配体。高亲和力配体引发转变而不改变封闭状态的寿命，而低亲和力配体则显着缩短寿命。我们表明，封闭状态下的 SBD 竞争与转运蛋白对接，但令人惊讶的是，在没有配体的情况下，效果最强。我们发现，决定步骤取决于 SBD 和转运的氨基酸。我们得出结论，封闭构象的寿命控制 SBD 对接转运蛋白和底物释放。
