Convergent MurJ flippase inhibition by phage lysis proteins

Antimicrobial drug resistance poses a global health challenge that necessitates the identification of new druggable targets^1-3. The essential lipid II flippase MurJ is a promising yet underexplored antimicrobial target in bacterial cell wall biosynthesis^4-7. The only known inhibitors of Gram-negative (diderm) MurJ are the single-gene lysis proteins (Sgls) from the lytic single-strand RNA phages M (Sgl^M) and PP7 (Sgl^PP7)^8,9. Sgl^M and Sgl^PP7 have distinct evolutionary origins and share no sequence similarity. Here we describe a common mechanism of MurJ inhibition by these phage-encoded Sgls. We determined the structures of MurJ-bound Sgl^M and Sgl^PP7 and discovered a third distinct MurJ-targeting Sgl from the predicted phage Changjiang3 (Sgl^CJ3) that we also characterized structurally. Our findings demonstrate that all three Sgls evolved convergently to trap MurJ in a periplasm-open conformation through a common MurJ interface, revealing a pathway for drug design.