Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity

Abstract

The antimicrobial peptide LL-37 belongs to the cathelicidin family and is the first amphipathic alpha-helical peptide isolated from human. LL-37 is considered to play an important role in the first line of defence against local infection and systemic invasion of pathogens at sites of inflammation and wounds. Understanding its mode of action may assist in the development of antimicrobial agents mimicking those of the human immune system. In vitro studies revealed that LL-37 is cytotoxic to both bacterial and normal eukaryotic cells. To gain insight into the mechanism of its non-cell-selective cytotoxicity, we synthesized and structurally and functionally characterized LL-37, its N-terminal truncated form FF-33, and their fluorescent derivatives (which retained structure and activity). The results showed several differences, between LL-37 and other native antimicrobial peptides, that may shed light on its in vivo activities. Most interestingly, LL-37 exists in equilibrium between monomers and oligomers in solution at very low concentrations. Also, it is significantly resistant to proteolytic degradation in solution, and when bound to both zwitterionic (mimicking mammalian membranes) and negatively charged membranes (mimicking bacterial membranes). The results also showed a role for the N-terminus in proteolytic resistance and haemolytic activity, but not in antimicrobial activity. The LL-37 mode of action with negatively charged membranes suggests a detergent-like effect via a 'carpet-like' mechanism. However, the ability of LL-37 to oligomerize in zwitterionic membranes might suggest the formation of a transmembrane pore in normal eukaryotic cells. To examine this possibility we used polarized attenuated total reflectance Fourier-transform infrared spectroscopy and found that the peptide is predominantly alpha-helical and oriented nearly parallel with the surface of zwitterionic-lipid membranes. This result does not support the channel-forming hypothesis, but rather it supports the detergent-like effect.

Amphipathic, α-helical antimicrobial peptides

“…It is possible that, under the influence of the anisotropic environment at the membrane surface, these peptides seek different conformations so as to maximize their amphipathicity, and thus achieve a somewhat different spectrum of activity. 192 It is thus apparent that the maximum potency in ␣-helical AMPs is obtained when a high charge and amphipathicity and increased structuring are achieved. Reduction of charge or depth of the hydrophobic sector results in a general decrease in potency, while reduction of amphipathicity and structuring results in a more selective reduction in antimicrobial activity.…”

Structural features and biological activities of the cathelicidin-derived antimicrobial peptides

“…22,27,28,46,78,81,82,85 Some of them, particularly those with C-terminal (BMAP-27, BMAP-28, and SMAP-29) or N-terminal (LL-37/ hCAP18) hydrophobic regions, are also toxic to eukaryotic cells, 22,81,84,85 while others, such as PMAP-23, CRAMP, and BMAP-34, are much less or not at all effective. 27,46,78 Targets include normal and tumor cell lines and red blood cells, 22,84 and the effect is often exerted at concentrations that are only slightly higher than those active against microorganisms.…”

“…37,82 Synthetic fragments of BMAP-27, BMAP-28, and LL-37/hCAP18, lacking their hydrophobic region, show an improved selectivity, with a significant reduction in cytotoxic activity to eukaryotic cells and an unchanged or only modestly decreased antibacterial activity. 22,84,85 The role of the hydrophobic region has further been investigated in BMAP-28. An analogue of this peptide, in which the highly hydrophobic residues at the C-terminus were replaced with more hydrophilic ones, shows a markedly reduced toxicity toward mammalian cells.…”

Nonperturbative Imaging of Nucleoid Morphology in Live Bacterial Cells during an Antimicrobial Peptide Attack

“…Cecropin A and LL-37 are similar in length (37 amino acids) and charge (ϩ7) and exist as amphipathic ␣-helices on binding to model lipid bilayers (21,22). We previously reported the sequence of events involved in the membrane permeabilization and growth inhibition caused by these peptides (19).…”