Department of Molecular Biology , Princeton University , Princeton , New Jersey 08544 , United States.
Howard Hughes Medical Institute , Chevy Chase , Maryland 20815 , United States.
ACS Chem Biol. 2019 Mar 15;14(3):378-389. doi: 10.1021/acschembio.8b00971. Epub 2019 Mar 4.
Bacteria use a cell-cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150°. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of LasR residues Thr75, Tyr93, and Ala127 converts low-potency compounds into high-potency compounds and converts ligands that are nearly inactive into low-potency compounds. These results show that the LasR binding pocket displays significant flexibility in accommodating different ligands. The ability of LasR to bind ligands in different conformations, and in so doing, alter their potency as agonists, could explain the difficulties that have been encountered in the development of competitive LasR inhibitors.
细菌使用一种称为群体感应的细胞间通讯过程来协调集体行为。群体感应依赖于细胞外信号分子（称为自诱导物）的产生和全组检测。在这里，我们使用基于天然同型丝氨酸内酯自诱导物结构的合成激动剂来探测铜绿假单胞菌 LasR 群体感应受体的活性。这些合成化合物的效力从低到高不等，激动剂活性与激动剂稳定 LasR 蛋白的能力相关。LasR 配体结合域与代表性合成激动剂复合物的结构分析揭示了两种配体结合模式，一种模拟经典的自诱导物结合排列，另一种带有内酯头基团旋转约 150°。迭代诱变结合化学合成分别揭示了分别使每种结合模式成为可能的氨基酸残基和化学部分。同时改变 LasR 残基 Thr75、Tyr93 和 Ala127 将低效力化合物转化为高效力化合物，并将几乎无活性的配体转化为低效力化合物。这些结果表明 LasR 结合口袋在容纳不同配体方面具有很大的灵活性。LasR 能够结合不同构象的配体，并且通过这种方式改变它们作为激动剂的效力，这可以解释在开发竞争性 LasR 抑制剂方面所遇到的困难。
