PDF(Pubmed)
Abstract：
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 Lactococcus lactis 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.
摘要：
细菌利用各种策略来防止高渗胁迫期间的内部脱水。应对压力影响的常见方法是导入兼容的溶质，如甘氨酸甜菜碱，导致渗透梯度后同时产生被动水通量。来自乳酸乳球菌的OpuA是I型ABC-导入体,其使用两个底物结合结构域(SBD)来捕获细胞外甘氨酸甜菜碱并将底物递送至跨膜结构域用于随后的转运。OpuA通过内部离子强度的变化来感知渗透压，并且还受到第二信使环di-AMP的调节。我们现在展示，通过基于溶液的单分子FRET和多参数逐光子隐马尔可夫模型分析，SBD以离子强度依赖的方式暂时相互作用。smFRET数据符合运输中的明显协同性，并得到OpuA的新低温EM数据的支持。我们认为SBD之间的物理相互作用和底物传递中的协同性是传输机制的一部分。
