Lipopolysaccharide-bound structure of the antimicrobial peptide cecropin P1 determined by nuclear magnetic resonance spectroscopy.

Antimicrobial peptides (AMPs) are components of the innate immune system and may be potential alternatives to conventional antibiotics because they exhibit broad-spectrum antimicrobial activity. The AMP cecropin P1 (CP1), isolated from nematodes found in the stomachs of pigs, is known to exhibit antimicrobial activity against Gram-negative bacteria. In this study, we investigated the interaction between CP1 and lipopolysaccharide (LPS), which is the main component of the outer membrane of Gram-negative bacteria, using circular dichroism (CD) and nuclear magnetic resonance (NMR). CD results showed that CP1 formed an α-helical structure in a solution containing LPS. For NMR experiments, we expressed (15) N-labeled and (13) C-labeled CP1 in bacterial cells and successfully assigned almost all backbone and side-chain proton resonance peaks of CP1 in water for transferred nuclear Overhauser effect (Tr-NOE) experiments in LPS. We performed (15) N-edited and (13) C-edited Tr-NOE spectroscopy for CP1 bound to LPS. Tr-NOE peaks were observed at the only C-terminal region of CP1 in LPS. The results of structure calculation indicated that the C-terminal region (Lys15-Gly29) formed the well-defined α-helical structure in LPS. Finally, the docking study revealed that Lys15/Lys16 interacted with phosphate at glucosamine I via an electrostatic interaction and that Ile22/Ile26 was in close proximity with the acyl chain of lipid A.