Key role of the loop connecting the two beta strands of mussel defensin in its antimicrobial activity.
To elucidate the structural features of the mussel defensin MGD1 required for antimicrobial activity, we synthesized a series of peptides corresponding to the main known secondary structures of the molecule and evaluated their activity towards Gram-positive and Gram-negative bacteria, and filamentous fungi. We found that the nonapeptide corresponding to residues 25-33 of MGD1 (CGGWHRLRC) exhibited bacteriostatic activity once it was cyclized by a non-naturally occurring disulfide bridge. Longer peptides corresponding to the amino acid sequences of the alpha-helical part or to the beta-strands of MGD1 had no detectable activity. The bacteriostatic activity of the sequence 25-33 was strictly dependent on the bridging of Cys25 and Cys33 and was proportional to the theoretical isoelectric point of the peptide, as deduced from the variation of activity in a set of peptide analogues of the 25-33 sequence with different numbers of cationic charges. By using confocal fluorescence microscopy, we found that the cyclic peptides bound to Gram-positive bacteria without apparent lysis. However, by using a fluorescent dye, we observed that dead bacteria had been permeated by the cyclic peptide 25-33. Sequence comparisons in the family of arthopod defensins indicate that MGD1 belongs to a subfamily of the insect defensins, characterized by the constant occurrence of both positively charged and hydrophobic amino acids in the loop. Modelling studies showed that in the MGD1 structure, positively charged and hydrophobic residues are organized in two layered clusters, which might have a functional significance in the docking of MGD1 to the bacterial membrane.
Romestand
Bernard
B
DRIM, Université Montpellier 2, France.
Molina
Franck
F
Richard
Véronique
V
Roch
Philippe
P
Granier
Claude
C
D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001419:Bacteria / Q000378:metabolism; D049872:Bivalvia / Q000737:chemistry; D023082:Defensins; D005670:Fusarium / Q000378:metabolism; D007526:Isoelectric Point; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D023181:Antimicrobial Cationic Peptides; D023082:Defensins; C101524:MGD-1 protein, Mytilus; D011506:Proteins