Plasticity in structure and interactions is critical for the action of indolicidin, an antibacterial peptide of innate immune origin

Abstract

The comparative analysis of two cationic antibacterial peptides of the cathelicidin family-indolicidin and tritrypticin-enabled addressing the structural features critical for the mechanism of indolicidin activity. Functional behavior of retro-indolicidin was found to be identical to that of native indolicidin. It is apparent that the gross conformational propensities associated with retro-peptides resemble those of the native sequences, suggesting that native and retro-peptides can have similar structures. Both the native and the retro-indolicidin show identical affinities while binding to endotoxin, the initial event associated with the antibacterial activity of cationic peptide antibiotics. The indolicidin-endotoxin binding was modeled by docking the indolicidin molecule in the endotoxin structure. The conformational flexibility associated with the indolicidin residues, as well as that of the fatty acid chains of endotoxin combined with the relatively strong structural interactions, such as ionic and hydrophobic, provide the basis for the endotoxin-peptide recognition. Thus, the key feature of the recognition between the cationic antibacterial peptides and endotoxin is the plasticity of molecular interactions, which may have been designed for the purpose of maintaining activity against a broad range of organisms, a hallmark of primitive host defense.

Fig. 1.

Comparison of tritrypticin analog Sym11 with indolicidin. (A) Antibacterial activity against Samonella typhimurium DVWYPYPYASGS is an unrelated peptide used as control. (B) Dose-dependent effect of MgCl₂ on the antibacterial activity of the peptides against S. typhimurium. (C) Competitive displacement of dansyl polymyxin B in a dose-dependent manner by different peptides for endotoxin binding. (D) Sequence comparisons involving Sym11, indolicidin (N-indo) and retro-indolicidin (R-indo).

Fig. 2.

Comparison of indolicidin with retro-indolicidin. (A) Antibacterial activity against Salmonella typhimurium. (B) Dose-dependent effect of MgCl₂ on the antibacterial activity of the peptides against S. typhimurium. (C) Competitive displacement of dansyl polymyxin B in a dose-dependent manner by different peptides for endotoxin binding.

Fig. 3.

Model of indolicidin–LPS binding. Stereoscopic diagram of the complex of LPS with indolicidin. LPS is shown as Connolly surface and is marked as follows: green, carbon; red, oxygen; blue, nitrogen; white, hydrogen; purple, phosphates. Indolicidin is shown in navy blue stick with residues marked.

Fig. 4.

LPS affinity measurements of (A) indolicidin and (B) retro-indolicidin. The sensogram representing the binding signal of the peptide to the immobilized LPS is shown as a function of peptide concentration. The inset shows the linear fit of the K_on with the peptide concentration.

Fig. 5.

Comparison of circular dichroism profiles of (A) indolicidin and (B) retro-indolicidin, in free and LPS-bound forms.

Fig. 6.

The D-analogs of indolicidin and retro-indolicidin show similar functional behavior as the corresponding L-peptides. Comparison of the antibacterial activities against Salmonella typhimurium of the two peptides is plotted in a dose-dependent manner.