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 2 O (1 : 1, v/v) solution has been determinedby NMR spectroscopy previously and consists of two amphipathic α-helices from Leu4 to LyslO and from Gly16 to Leu33. These two helices are connected by a flexible region from Glyl 1 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 1 4 , 1 5 → 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 2 O (1: 1, v: v) solution shows amphipathic α-helix, from Glu 2 to Leu 1 8 , while CRAMP-18-A has relatively short amphipathic α-helix from Leu 4 to Ala 1 5 . These results suggest that the hydrophobic property of Phe 1 4 and Phe 1 5 in CRAMP-18 is essential for its antibacterial activity, α-helical structure, and interactions with phospholipid membranes.