Interactions of the Novel Antimicrobial Peptide Buforin 2 with Lipid Bilayers:Proline as a Translocation Promoting Factor

摘要

Buforin 2 is an antimicrobial peptide discovered inthe stomach tissue of the Asian toad Bufo bufo gargarizans. The 21-residue peptide with +6 net charge shows antimicrobial activity an order of magnifude higher than that of magainin 2, a membrane-permeabilizing antimicrobial peptide from Xenopus laevis [Park, C.B.,Kim, M.S.,and Kim,S.C.(1996) Biochem. Biophys. Res.Commun.218, 408-413]. In this study, we investigated the interactions of buforin 2 withphospholipid bilayers in comparison with magainin 2 to obtaiin insight into the mechanism of action of buforin 2. Equipotent Trp-substituted peptides were used to fluormetrically monitor peptide-lipid interactions. Circular dchroism measurements showed that buforin 2 selectively bound to liposomes composed of acidic phospholipids, assuming a secondary structure similar to that in trifluoroethanol/water, which is an amphipathic helix dstorted around Pro~(11) with a flexible N-terminal region [Yi, G.S.,Park, C.B.,Kim,S.B.,Kim, S.C.,and Cheong, C.(1996) FEBS Lett. 398, 87-90]. Magainin 2 induced the leakage of a fluorescent dye entrapped within lipid vesicles coupled to lipid flip-flop. These results have been interpreted as the formation of a peptide-lipid supramolecular complex pore [Matsuzaki,K.(1998) Biochim. Biophys Acta1376,391-400]. Buforin 2 exhibited much weaker membrane permeabilization activity despite its higher antimicrobial activity. In contrast,buforin 2 was more efficiently transolocated across lipid bilayers than magainin 2. These results suggested that the ultimate target of buforin 2 is not membrane but intracellular components. Futhermore, buforin 2 induced no lipid flip-flop, indicating that the mechanism of translocation of buforin 2 is different from that of magainin 2. The role of Pro was investigated by use of a P11A derivative of buforin 2. The derivation caused a change to magainin 2-like secondayr structure and membrane behavior. Pro~(11) was found to be a very important structural factor for the unique properties of buforin 2.