Department of Chemistry, Chemistry Research Laboratory, University of Oxford , Mansfield Road, Oxford OX1 3TA, United Kingdom.
Biochemistry. 2014 Apr 1;53(12):1982-92. doi: 10.1021/bi5001118. Epub 2014 Mar 21.
The potassium efflux system, Kef, protects bacteria against the detrimental effects of electrophilic compounds via acidification of the cytoplasm. Kef is inhibited by glutathione (GSH) but activated by glutathione-S-conjugates (GS-X) formed in the presence of electrophiles. GSH and GS-X bind to overlapping sites on Kef, which are located in a cytosolic regulatory domain. The central paradox of this activation mechanism is that GSH is abundant in cells (at concentrations of ∼10-20 mM), and thus, activating ligands must possess a high differential over GSH in their affinity for Kef. To investigate the structural requirements for binding of a ligand to Kef, a novel fluorescent reporter ligand, S-{[5-(dimethylamino)naphthalen-1-yl]sulfonylaminopropyl} glutathione (DNGSH), was synthesized. By competition assays using DNGSH, complemented by direct binding assays and thermal shift measurements, we show that the well-characterized Kef activator, N-ethylsuccinimido-S-glutathione, has a 10-20-fold higher affinity for Kef than GSH. In contrast, another native ligand that is a poor activator, S-lactoylglutathione, exhibits a similar Kef affinity to GSH. Synthetic ligands were synthesized to contain either rigid or flexible structures and investigated as ligands for Kef. Compounds with rigid structures and high affinity activated Kef. In contrast, flexible ligands with similar binding affinities did not activate Kef. These data provide insight into the structural requirements for Kef gating, paving the way for the development of a screen for potential therapeutic lead compounds targeting the Kef system.
钾外排系统（Kef）通过使细胞质酸化来保护细菌免受亲电化合物的有害影响。谷胱甘肽（GSH）抑制 Kef，但在亲电试剂存在下形成的谷胱甘肽-S-共轭物（GS-X）可激活 Kef。GSH 和 GS-X 结合到位于细胞质调节域上的 Kef 的重叠结合位点上。该激活机制的核心悖论是细胞中 GSH 含量丰富（浓度约为 10-20mM），因此，激活配体必须具有比 GSH 更高的对 Kef 的亲和力差异。为了研究配体与 Kef 结合的结构要求，合成了一种新型荧光报告配体 S-[[5-(二甲基氨基)萘-1-基]磺酰基氨基丙基]谷胱甘肽（DNGSH）。通过使用 DNGSH 的竞争测定，辅以直接结合测定和热移位测量，我们表明，经过充分表征的 Kef 激活剂 N-乙基琥珀酰亚胺-S-谷胱甘肽对 Kef 的亲和力比 GSH 高 10-20 倍。相比之下，另一种作为不良激活剂的天然配体 S-乳酰谷胱甘肽对 Kef 的亲和力与 GSH 相似。合成的配体被设计成具有刚性或柔性结构，并作为 Kef 的配体进行研究。具有刚性结构和高亲和力的化合物可激活 Kef。相比之下，具有相似结合亲和力的柔性配体则不能激活 Kef。这些数据提供了对 Kef 门控结构要求的深入了解，为开发针对 Kef 系统的潜在治疗先导化合物筛选铺平了道路。
