Structural basis for antibiotics murepavadin and thanatin targeting the lipopolysaccharide insertase LptD

Gram-negative bacteria are shielded by an asymmetric outer membrane whose biogenesis depends on the lipopolysaccharide transport (Lpt) machinery. Therein, the essential membrane protein LptD mediates the final lipopolysaccharide insertion step, making it a prime target for antibiotics. Here, we determined five high-resolution cryo-electron microscopy structures of Escherichia coli and Pseudomonas aeruginosa LptDEM in complex with the peptide antibiotics murepavadin and thanatin, to resolve their modes of action. The structures reveal that both compounds target the distal β-jellyroll edge of LptD, blocking the essential interaction with the periplasmic bridge protein LptA. Despite converging on the same binding region, thanatin and murepavadin engage LptD via different interaction modes: thanatin binds via β-sheet augmentation, whereas murepavadin binds through side chain interactions. These alternative strategies explain the broad-spectrum activity of thanatin versus the species specificity of murepavadin and establish the distal β-jellyroll edge of LptD as a structural vulnerability in the Gram-negative envelope. Our findings thus provide a framework for rational design of antibiotics targeting this site.