Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 2018 Jun 26;115(26):6709-6714. doi: 10.1073/pnas.1802192115. Epub 2018 Jun 11.
The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein before its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data for MurJ are available only from the extremophile Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organism , revealing an inward-open conformation. Taking advantage of the genetic tractability of , we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Lastly, through the use of sequence coevolution analysis, we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.
肽聚糖细胞壁为几乎所有细菌提供了必不可少的保护屏障，定义了细胞形态，并赋予其抵抗渗透压和其他环境危害的能力。肽聚糖的前体脂质 II 在内膜层的质膜上组装。然而，肽聚糖的聚合发生在质膜的外表面，并且在将脂质 II 用于肽聚糖合成之前，MurJ 蛋白必须将其翻转穿过膜。由于 MurJ 在细胞壁组装中的核心作用，它在微生物细胞生物学中具有至关重要的作用，是新型抗生素开发的主要目标。然而，关于 MurJ 功能的机制相对知之甚少，并且仅从极端微生物中获得了 MurJ 的结构数据。在这里，我们报告了革兰氏阴性模式生物 的无底物 MurJ 的晶体结构，揭示了一种向内开放的构象。利用 的遗传可操作性，我们进行了高通量诱变和下一代测序，以评估蛋白质中每个氨基酸的突变耐受性，为该酶提供了详细的功能和结构图谱，并确定了抑制剂开发的位点。最后，通过序列共进化分析，我们确定了蛋白质外向开放状态下的功能重要相互作用，支持脂质 II 转运的摇臂开关模型。
