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 <i>Escherichia coli</i>, MurJ, the lipid II flippase, is an essential membrane protein. The 37-residue protein M from the <i>Levivirus</i> 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-angstrom 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.