Department of Chemistry and Chemical Biology , Harvard University , Cambridge , Massachusetts 02138 , United States.
Department of Microbiology , The Ohio State University , Columbus , Ohio 43210 , United States.
J Am Chem Soc. 2018 Apr 4;140(13):4481-4484. doi: 10.1021/jacs.8b00942. Epub 2018 Mar 26.
MurJ, the flippase that exports the bacterial cell wall monomer Lipid II to the periplasm, is a target for new antibiotics, which are desperately needed to treat Gram-negative infections. Quantitative methods to monitor MurJ activity are required to characterize inhibitors but are challenging to develop because the lipid-linked substrate is not chemically altered in a flippase reaction. Here we show that MurJ inhibition can be quantified by measuring the accumulation of intracellular Lipid II using a biotin-tagging strategy. We have exploited this assay to show that MurJ is inhibited in the presence of a compound that dissipates the membrane potential. By probing cysteine accessibility we have found that under this condition MurJ relaxes into an inactive, outward-facing conformation reminiscent of that targeted by the peptide antibiotic Lys. We conclude that membrane potential is required for MurJ function in E. coli, and we anticipate that the ability to accumulate this inactive conformation will lead to structures useful for inhibitor design.
MurJ 是一种翻转酶，可将细菌细胞壁单体 Lipid II 输出到周质空间，是新型抗生素的靶标，而新型抗生素对于治疗革兰氏阴性菌感染是急需的。为了表征抑制剂，需要定量方法来监测 MurJ 活性，但由于翻转酶反应中脂质连接的底物没有发生化学变化，因此开发起来具有挑战性。在这里，我们展示了可以通过使用生物素标记策略测量细胞内 Lipid II 的积累来定量 MurJ 抑制作用。我们利用该测定法表明，在存在一种使膜电位耗散的化合物的情况下，MurJ 被抑制。通过探测半胱氨酸可及性，我们发现在此条件下，MurJ 松弛成一种无活性的外向构象，类似于肽抗生素 Lys 靶向的构象。我们得出结论，膜电位是 MurJ 在大肠杆菌中发挥功能所必需的，并且我们预计能够积累这种无活性构象的能力将产生对抑制剂设计有用的结构。
