Crystal structure of the lipid flippase MurJ in a “squeezed” form distinct from its inward- and outward-facing forms

• Published in: Structure (London) 2022-08, Vol.30 (8), p.1088-1097.e3
• Main Authors: Kohga, Hidetaka, Mori, Takaharu, Tanaka, Yoshiki, Yoshikaie, Kunihito, Taniguchi, Katsuhide, Fujimoto, Kei, Fritz, Lisa, Schneider, Tanja, Tsukazaki, Tomoya
• Format: Article
• Language: English
• Subjects: Lipid II flippase; MD simulation; membrane protein; MurJ; transporter; X-ray crystallography
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2022.05.008

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. [Display omitted] •Crystal structures of bacterial MurJ reveal its squeezed and inward forms•The squeezed form does not contain an internal cavity required for substrate binding•Transmembrane helices 2, 7, and 8 contribute to MurJ structural transitions•The squeezed form occurs during outward-to-inward conformational changes Kohga et al. present an unprecedented “squeezed” structure of MurJ, a Lipid II flippase that is essential for peptidoglycan biogenesis in gram-negative bacteria. The “squeezed” form is proposed to be an intermediate during outward-to-inward conformational changes after substrate release, which is further supported by molecular dynamics simulations.