Role of the hinge region and the tryptophan residue in the synthetic antimicrobial peptides, cecropin A(1-8)-magainin 2(1-12) and its analogues, on their antibiotic activities and structures

A 20-residue hybrid peptide CA(1-8)-MA(1-12) (CA-MA), incorporating residue s 1-8 of cecropin A (CA) and residues 1-12 of magainin 2 (MA), has potent a ntimicrobial activity without toxicity against human erythrocytes. To inves tigate the effects of the Gly-Ile-Gly hinge sequence of CA-MA on the antiba cterial and antitumor activities, two analogues in which the Gly-Ile-Gly se quence of CA-MA is either deleted (P1) or substituted with Pro (P2) were sy nthesized. The role of the tryptophan residue at position 2 of CA-MA on its antibiotic activity was also investigated using two analogues, in which th e Trp2 residue of CA-MA is replaced with either Ala (P3) or Leu (P4). The t ertiary structures of CA-MA, P2, and P4 in DPC micelles, as determined by N MR spectroscopy, have a short amphiphilic helix in the N-terminus and about three turns of alpha-helix in the C-terminus, with the flexible hinge regi on between them. The P1 analogue has an alpha-helix from Leu4 to Ala14 with out any hinge structure. P1 has significantly decreased lyric activities ag ainst bacterial and tumor cells and PC/PS vesicles (3:1, w/w), and reduced pore-forming activity on lipid bilayers, while P2 retained effective lytic activities and pore-forming activity. The N-terminal region of P3 has a fle xible structure without any specific secondary structure, The P3 modificati on caused a drastic decrease in the antibiotic activities, whereas P4, with the hydrophobic Leu side chain at position 2, retained its activities. On the basis of the tertiary structures, antibiotic activities, vesicle-disrup ting activities, and pore-forming activities, the structure-function relati onships can be summarized as follows. The partial insertion of the Trp2 of CA-MA into the membrane, as well as the electrostatic interactions between the positively charged Lys residues at the N-terminus of the CA-MA and the anionic phospholipid headgroups, leads to the primary binding to the cell m embrane. Then, the flexibility or bending potential induced by the Gly-Ile- Gly hinge sequence or the Pro residue in the central part of the peptides m ay allow the alpha-helix in the C-terminus to span the lipid bilayer. These structural features are crucial for the potent antibiotic activities of CA -MA.