The structure of the lipid II flippase from monoderm bacteria

Peptidoglycan biogenesis requires membrane flippases to translocate lipid-linked precursors across the cytoplasmic membrane for processing (1). This essential step is mediated by MurJ, the lipid II flippase conserved across all peptidoglycan-producing bacteria (2). While MurJ from diderm bacteria has been structurally resolved in multiple conformational states (3-6), its monoderm homolog remains uncharacterized. Monoderm MurJ homologs exhibit substantial sequence divergence yet retain the same lipid II flipping function (7) and are promising antibiotic targets. Here we report structures of Staphylococcus aureus MurJ ( Sa MurJ) captured in both outward- and inward-facing conformations. These structures show that Sa MurJ adopts the conserved MOP family fold and undergoes conformational transitions consistent with an alternating-access mechanism. Our findings reveal conserved and divergent features of MurJ between diderm and monoderm bacteria that are critical for lipid II flipping and provide a structural framework for probing substrate recognition and specific inhibition.

The growing global threat of antibiotic resistance and the limited development of new antibacterial therapies underscore the urgent need to identify and mechanistically characterize new antibiotic targets and mechanisms. MurJ is an essential membrane transporter required for cell wall biosynthesis and represents an attractive but unexplored antibiotic target. Here we determine the structures of MurJ from a clinically critical monoderm pathogen Staphylococcus aureus in key conformational states during its transport cycle. This work advances our understanding of an essential step in bacterial cell wall synthesis, reveals key distinctions between monoderm and diderm MurJ, and defines structural features that can be exploited for antibiotic discovery.