Understanding the structural requirements for activators of the Kef bacterial potassium efflux system.
Understanding the structural requirements for activators of the Kef bacterial potassium efflux system.
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DOI：
10.1021/bi5001118
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发表时间：
2014-04-01
期刊：
影响因子：
2.9
通讯作者：
Rasmussen T
中科院分区：
文献类型：
--
作者：
Healy J;Ekkerman S;Pliotas C;Richard M;Bartlett W;Grayer SC;Morris GM;Miller S;Booth IR;Conway SJ;Rasmussen T
钾外排系统，Kef，通过细胞质酸化保护细菌免受亲电化合物的有害影响。Kef被谷胱甘肽（GSH）抑制，但被在亲电试剂存在下形成的谷胱甘肽- s共轭物（GS-X）激活。GSH和GS-X结合到Kef上的重叠位点，这些位点位于细胞质调节区域。这种激活机制的核心矛盾在于细胞中谷胱甘肽含量丰富（浓度为~ 10-20 mM），因此，激活配体对Kef的亲和力必须比谷胱甘肽高。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-（二甲氨基）萘-1-基]磺酰氨基丙基}谷胱甘肽（DNGSH）。通过使用DNGSH的竞争实验，结合直接结合实验和热位移测量，我们发现表征良好的Kef激活剂n -乙基琥珀酰氨基- s -谷胱甘肽对Kef的亲和力比谷胱甘肽高10 - 20倍。相比之下，另一种天然配体s -乳酸谷胱甘肽是一种较差的激活剂，对谷胱甘肽表现出类似的Kef亲和力。合成了具有刚性或柔性结构的配体，并对其作为Kef的配体进行了研究。具有刚性结构和高亲和力的化合物活化Kef。相反，具有相似结合亲和力的柔性配体不会激活Kef。这些数据提供了对Kef门控的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的筛选铺平了道路。
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.
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DOI：
10.1016/j.str.2009.03.018
发表时间：
2009-06-10
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Roosild TP;Castronovo S;Miller S;Li C;Rasmussen T;Bartlett W;Gunasekera B;Choe S;Booth IR
通讯作者：
Booth IR
影响因子：
3.6
作者：
FERGUSON, GP;MCLAGGAN, D;BOOTH, IR
通讯作者：
BOOTH, IR
影响因子：
3.2
作者：
EPSTEIN, W;KIM, BS
通讯作者：
KIM, BS
影响因子：
4.4
作者：
McLaggan, D;Rufino, H;Booth, IR
通讯作者：
Booth, IR
影响因子：
3.9
作者：
CHEN, RF
通讯作者：
CHEN, RF