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,

Abstract

A 20-residue hybrid peptide CA(1−8)−MA(1−12) (CA−MA), incorporating residues 1−8 of cecropin A (CA) and residues 1−12 of magainin 2 (MA), has potent antimicrobial activity without toxicity against human erythrocytes. To investigate the effects of the Gly-Ile-Gly hinge sequence of CA−MA on the antibacterial and antitumor activities, two analogues in which the Gly-Ile-Gly sequence of CA−MA is either deleted (P1) or substituted with Pro (P2) were synthesized. The role of the tryptophan residue at position 2 of CA−MA on its antibiotic activity was also investigated using two analogues, in which the Trp2 residue of CA−MA is replaced with either Ala (P3) or Leu (P4). The tertiary structures of CA−MA, P2, and P4 in DPC micelles, as determined by NMR spectroscopy, have a short amphiphilic helix in the N-terminus and about three turns of α-helix in the C-terminus, with the flexible hinge region between them. The P1 analogue has an α-helix from Leu4 to Ala14 without any hinge structure. P1 has significantly decreased lytic activities against 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 flexible structure without any specific secondary structure. The P3 modification 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-disrupting activities, and pore-forming activities, the structure−function relationships 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 membrane. 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 may allow the α-helix in the C-terminus to span the lipid bilayer. These structural features are crucial for the potent antibiotic activities of CA−MA.