Structural and Functional Characterization of CRAMP-18 Derived from a Cathelicidin-Related Antimicrobial Peptide CRAMP

CRAMP was identified from a cDNA clone derived from a mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptide. Tertiary structure of CRAMP in TFE/$H_2O$ (1 : 1, v/v) solution has been determined by NMR spectroscopy previously and consists of two amphipathic $\alpha-helices$ from Leu4 to Lys10 and from Gly16 to Leu33. These two helices are connected by a flexible region from Gly11 to Gly16. Analysis of series of fragments composed of various portion of CRAMP revealed that an 18-residue fragment with the sequence from Gly16 to Leu33 (CRAMP-18) was found to retain antibacterial activity without cytotoxicity. The effects of two Phe residues at positions 14 and 15 of CRAMP-18 on structure, antibacterial activity, and interaction with lipid membranes were investigated by $Phe^{14,15}$ ${\rightarrow}$ Ala substitution (CRAMP-18-A) in the present study. Substitution of Phe with Ala in CRAMP-18 caused a significant reduction on antibacterial and membrane-disrupting activities. Tertiary structures of CRAMP-18 in 50% TFE/$H_2O$ (1 : 1, v : v) solution shows amphipathic ${\alpha}$-helix, from $Glu^2{\;}to{\;}Leu^{18}$, while CRAMP-18-A has relatively short amphipathic ${\alpha}$-helix from $Leu^4{\;}to{\;}Ala^{15}$. These results suggest that the hydrophobic property of $Phe^{14}{\;}and{\;}Phe^15$ in CRAMP-18 is essential for its antibacterial activity, ${\alpha}$-helical structure, and interactions with phospholipid membranes.