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 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 alpha-helix in the C-terminus, with the flexible hinge region between them. The P1 analogue has an alpha-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 alpha-helix in the C-terminus to span the lipid bilayer. These structural features are crucial for the potent antibiotic activities of CA-MA.
Oh
D
D
Department of Chemistry, Konkuk University, Seoul 143-701, Korea.
Shin
S Y
SY
Lee
S
S
Kang
J H
JH
Kim
S D
SD
Ryu
P D
PD
Hahm
K S
KS
Kim
Y
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000138:chemical synthesis* / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000138:chemical synthesis* / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects / Q000254:growth & development; D004553:Electric Conductivity; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development; D006801:Humans; D007473:Ion Channels / Q000737:chemistry; D019169:Jurkat Cells; D020014:K562 Cells; D008051:Lipid Bilayers / Q000737:chemistry; D054806:Magainins; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000737:chemistry*; D029867:Xenopus Proteins
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D000970:Antineoplastic Agents; D007473:Ion Channels; D008051:Lipid Bilayers; D054806:Magainins; D010446:Peptide Fragments; D029867:Xenopus Proteins; C053301:magainin 2 peptide, Xenopus; C031161:cecropin A; D014364:Tryptophan