The bacterial peptidoglycan enclosing the cytoplasmic membrane is a fundamental cellular architecture. The integral membrane protein MurJ plays an essential role in flipping the cell wall building block Lipid II across the cytoplasmic membrane for peptidoglycan biosynthesis. Previously reported crystal structures of MurJ have elucidated its V-shaped inward- or outward-facing forms with an internal cavity for substrate binding. MurJ transports Lipid II using its cavity through conformational transitions between these two forms. Here, we report two crystal structures of inward-facing forms from Arsenophonus endosymbiont MurJ and an unprecedented crystal structure of Escherichia coli MurJ in a “squeezed” form, which lacks a cavity to accommodate the substrate, mainly because of the increased proximity of transmembrane helices 2 and 8. Subsequent molecular dynamics simulations supported the hypothesis that the squeezed form is an intermediate conformation. This study fills a gap in our understanding of the Lipid II flipping mechanism.

包围细胞质膜的细菌肽聚糖是一种基本的细胞结构。整合膜蛋白 MurJ 在将细胞壁结构单元脂质 II 翻转穿过细胞质膜以进行肽聚糖生物合成方面发挥着重要作用。先前报道的 MurJ 晶体结构已经阐明了其 V 形向内或向外的形式，具有用于底物结合的内腔。MurJ 通过这两种形式之间的构象转换利用其腔体运输脂质 II。在这里，我们报告了来自Arsenophonus endosymbiont MurJ 的两种内向形式的晶体结构和前所未有的大肠杆菌晶体结构MurJ 呈“挤压”形式，缺乏容纳底物的空腔，主要是因为跨膜螺旋 2 和 8 的接近度增加。随后的分子动力学模拟支持了挤压形式是中间构象的假设。这项研究填补了我们对脂质 II 翻转机制理解的空白。