Structures, Interactions and Activity of the N-Terminal Truncated Variants of Antimicrobial Peptide Thanatin

Gram-negative bacteria are intrinsically more resistant to many frontline antibiotics, which is attributed to the permeability barrier of the outer membrane, drug efflux pumps and porins. Consequently, discovery of new small molecules antibiotics to kill drug-resistant Gram-negative bacteria presents a significant challenge. Thanatin, a 21-residue insect-derived antimicrobial peptide, is known for its potent activity against Enterobacter Gram-negative bacteria, including drug-resistant strains. Here, we investigated a 15-residue N-terminal truncated analog PM15 (P¹IIYCNRRTGKCQRM¹⁵) of thanatin to determine modes of action and antibacterial activity. PM15 and the P¹ to Y and A substituted variants PM15Y and PM15A delineated interactions and permeabilization of the LPS-outer membrane. In antibacterial assays, PM15 and the analogs showed growth inhibition of strains of Gram-negative bacteria that is largely dependent on the composition of the culture media. Atomic-resolution structures of PM15 and PM15Y in free solution and in complex with LPS micelle exhibited persistent β-hairpin structures similar to native thanatin. However, in complex with LPS, the structures of peptides are more compact, with extensive packing interactions among residues across the two anti-parallel strands of the β-hairpin. The docked complex of PM15/LPS revealed a parallel orientation of the peptide that may be sustained by potential ionic and van der Waals interactions with the lipid A moiety of LPS. Further, PM15 and PM15Y bind to LptA_m, a monomeric functional variant of LptA, the periplasmic component of the seven-protein (A-G) complex involved in LPS transport. Taken together, the structures, target interactions and antibacterial effect of PM15 presented in the current study could be useful in designing thanatin-based peptide analogs.