Abstract
Cecropin P1 (CP1) isolated from a large roundworm Ascaris suum, which is found in pig intestines, has been extensively studied as a model antimicrobial peptide (AMP). However, despite being a model AMP, its antibacterial mechanism is not well understood, particularly the function of its C-terminus. By using an Escherichia coli overexpression system with calmodulin as a fusion partner, we succeeded in the mass expression of recombinant peptides, avoiding toxicity to the host and degradation of CP1. The structure of the recombinant (15)N- and (13)C-labeled CP1 and its C-terminus truncated analogue in dodecylphosphocholine (DPC) micelles was determined by NMR. In this membrane-mimetic environment, CP1 formed an α-helix for almost its entire length, except for a short region at the C-terminus, and there was no evidence of a hinge, which is considered important for the expression of activity in other cecropins. Several NMR analyses showed that the entire length of CP1 was protected from water by micelles. Since the loss of the C-terminus of the analogue had little effect on the NMR structure or its interaction with the micelle, we investigated another role of the C-terminus of CP1 in its antimicrobial activity. The results showed that the C-terminal region affected the DNA-binding capacity of CP1, and this mechanism of action was also newly suggested that it contributed to the antimicrobial activity of CP1.