Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Groningen, Netherlands.
Biochemistry Center, Heidelberg University, Heidelberg, Germany.
Elife. 2024 May 2;12:RP90996. doi: 10.7554/eLife.90996.
Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.
细菌利用各种策略来防止在高渗胁迫下内部脱水。一种常见的应对压力的方法是导入相容溶质，如甘氨酸甜菜碱，导致在渗透压梯度下同时发生被动水通量。来自 的 OpuA 是一种 I 型 ABC 转运体，它使用两个底物结合域（SBD）来捕获细胞外甘氨酸甜菜碱，并将底物递送到跨膜域进行后续运输。OpuA 通过内部离子强度的变化来感知渗透压胁迫，并且还受到 2 信使环二腺苷酸的调节。我们现在通过基于溶液的单分子 FRET 并使用多参数逐光子隐马尔可夫建模进行分析，表明 SBD 以离子强度依赖的方式瞬时相互作用。smFRET 数据与转运中的表观协同性一致，并得到 OpuA 的新冷冻电镜数据的支持。我们提出，SBD 之间的物理相互作用和底物传递中的协同性是运输机制的一部分。
