Phage lysis protein Lys ^M acts as a wedge to block MurJ conformational changes

Many antibiotics target essential cellular processes. To combat multidrug-resistant bacteria, new antibacterial strategies are needed. In the peptidoglycan biogenesis pathway in Escherichia coli , MurJ, the lipid II flippase, is an essential membrane protein. The 37-residue protein M from the Levivirus phage, known as Lys ^M or Sgl ^M , targets MurJ and induces cell lysis; however, its molecular mechanism remains unclear. Here, we present the cryo-EM structure of the MurJ/Lys ^M (JM) complex at 3.09 Å resolution, revealing that Lys ^M interacts with the crevasse between TM2 and TM7 of MurJ, locking MurJ in an outward-facing conformation, with Lys ^M acting like a wedge. Alanine-scanning mutagenesis and pull-down assays revealed key residues responsible for Lys ^M function, and molecular dynamics simulations showed that Lys ^M stabilizes MurJ’s outward-facing state. These findings demonstrate an unprecedented phage-derived mechanism for blocking lipid II transport, providing a structural framework for designing MurJ-targeted antimicrobial agents.