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Indolicidin, ILPWKWPWWPWRR-NH(2), a short 13-residue antimicrobial and cytolytic peptide characterized from bovine neutrophils, has the calmodulin-recognition 1-5-10 hydrophobic pattern (indicated by amino acids in bold), is cationic, and thereby fulfills the requirements to interact with calmodulin. Hence, we have investigated the calmodulin-binding properties of indolicidin. Indolicidin interacted with calmodulin with fairly high affinity in a Ca(2+)-dependent manner. However, when bound, the peptide did not adopt helical conformation. Indolicidin also inhibited calmodulin-stimulated phosphodiesterase activity with IC(50) values in the nanomolar range. Replacement of either the proline residues of indolicidin with alanines or tryptophan residues with phenylalanines did not affect binding to calmodulin. However, these replacements had distinctive effects on the conformations of the bound peptides. While the alanine analog of indolicidin adopted predominantly alpha-helical conformation, the phenylalanine analog remained largely unordered. Differences in the ability of these analogs to inhibit the calmodulin-stimulated phosphodiesterase activity were observed. While the alanine analog was capable of inhibiting the activity with IC(50) values comparable to that of indolicidin, the phenylalanine analog did not inhibit the activity. Our results indicate that ability to adopt amphiphilic alpha-helical structure is not a prerequisite for binding to calmodulin and also binding does not necessarily result in inhibition of calmodulin-stimulated enzyme activities.

The structure and biological activities of analogs of the bovine neutrophil antibacterial and hemolytic peptide indolicidin, ILPWKWPWWPWRR-amide, where one tryptophan at 4th, 8th, or 11th position has been retained and the others replaced by leucine, have been investigated. All the single tryptophan analogs exhibit antibacterial activity. However, unlike indolicidin, they do not lyse erythrocytes. Structure analysis by circular dichroism spectroscopy indicates that the analogs are unordered in aqueous medium and adopt beta-turn structures in trifluoroethanol and micelles. The tryptophan residues in indolicidin appear to be essential for hemolytic activity but not antibacterial activity. The nonspecific biological activities of indolicidin and specific antibacterial activity of single tryptophan analogs suggest that in short peptides, a motif composed of hydrophobic amino acids with the exception of tryptophan, interspaced with proline residues and cationic amino acids at the N or C termini would favor selective antibacterial activity., (Copyright 2000 Academic Press.)

Indolicidin, a 13-residue antimicrobial peptide isolated from cytoplasmic granules of bovine neutrophils, exhibits activity against Gram-positive and Gram-negative bacteria as well as fungi. Although indolicidin is bactericidal and permeabilizes the bacterial membranes, it does not lyse the bacterial cells. We examined the effect of bactericidal concentrations of indolicidin on the morphology of Escherichia coli cells and found that it induces filamentation. Further investigations showed that indolicidin inhibits DNA synthesis in E. coli cells at concentrations at which RNA and protein synthesis are either partially affected or not affected at all. Since inhibition of DNA synthesis is also known to induce filamentation in E. coli, it appears to contribute to the antimicrobial activity of indolicidin.

The antimicrobial and hemolytic activities of the 13-residue peptide indolicidin (ILPWKWPWWPWRR-NH2), present in bovine neutrophils, and its analogs have been determined with a view to gaining insight into the structural roles of tryptophan and proline. Peptides where proline was replaced by alanine and tryptophan by phenylalanine showed antibacterial activities comparable to that of indolicidin. The peptides do not exhibit a strong propensity to occur in either helical or beta-sheet conformation. The peptides also do not appear to exert their activity by permeabilizing the bacterial plasma membrane unlike other endogenous antibacterial peptides. The presence of tryptophan appears to be essential for hemolytic activity as the phenylalanine analog does not exhibit any hemolytic activity.