New aspects of the structure and mode of action of the human cathelicidin LL-37 revealed by the intrinsic probe p-cyanophenylalanine

Abstract

The human cathelicidin peptide LL-37 is an important effector of our innate immune system and contributes to host defence with direct antimicrobial activity and immunomodulatory properties, and by stimulating wound healing. Its sequence has evolved to confer specific structural characteristics that strongly affect these biological activities, and differentiate it from orthologues of other primate species. In the present paper we report a detailed study of the folding and self-assembly of this peptide in comparison with rhesus monkey peptide RL-37, taking into account the different stages of its trajectory from bulk solution to contact with, and insertion into, biological membranes. Phenylalanine residues in different positions throughout the native sequences of LL-37 and RL-37 were systematically replaced with the non-invasive fluorescent and IR probe p-cyanophenylalanine. Steady-state and time-resolved fluorescence studies showed that LL-37, in contrast to RL-37, forms oligomers with a loose hydrophobic core in physiological solutions, which persist in the presence of biological membranes. Fourier transform IR and surface plasmon resonance studies also indicated different modes of interaction for LL-37 and RL-37 with anionic and neutral membranes. This correlated with a distinctly different mode of bacterial membrane permeabilization, as determined using a flow cytometric method involving impermeant fluorescent dyes linked to polymers of defined sizes.

Membrane Core-Specific Antimicrobial Action of Cathelicidin LL-37 Peptide Switches Between Pore and Nanofibre Formation

“…The presence of an isodichroic point (202–204 nm) indicates two state helix-coil equilibrium32. The α-helical content for LL-37 in PBS buffer was estimated to be 34%, in good agreement with previous reports43. The helical conformation of LL-37 is increased upon binding of the peptide to the membranes (Table 1), which is common for lipid-binding peptides42.…”

The random co-polymer glatiramer acetate rapidly kills primary human leukocytes through sialic-acid-dependent cell membrane damage

“…Several studies have now clearly demonstrated the ability of LL-37 to form oligomers with a loose hydrophobic core under physiological conditions (37,56,57). Apparently, this oligomerization, or the character of peptides able to form such structures, is critical to the function of peptides of the LL-37 group, more so than specific size, sequence, or charge distribution (56).…”

Perspectives for clinical use of engineered human host defense antimicrobial peptides

“…Finally, the main direct mechanisms of action proposed for the cathelicidin LL-37 have been the bacterial membrane disruption following the toroidal pore model previously commented on and the probable interference with internal targets such as DNA (Xhindoli et al. 2015). …”