Conformation-dependent Antibacterial Activity of the Naturally Occurring Human Peptide LL-37

Abstract

The influence of ion composition, pH, and peptide concentration on the conformation and activity of the 37-residue human antibacterial peptide LL-37 has been studied. At micromolar concentration in water, LL-37 exhibits a circular dichroism spectrum consistent with a disordered structure. The addition of 15 mM HCO 3 ؊ , SO 4 2؊ , or CF 3 CO 2 ؊ causes the peptide to adopt a helical structure, with approximately equal efficiency, while 160 mM Cl ؊ is less efficient. A cooperative transition from disordered to helical structure is observed as the peptide concentration is increased, consistent with formation of an oligomer. The extent of ␣-helicity correlates with the antibacterial activity of LL-37 against both Gram-positive and Gram-negative bacteria. Two homologous peptides, FF-33 and SK-29, containing 4 and 8 residue deletions at the N terminus, respectively, require higher concentrations of anions for helix formation and are less active than LL-37 against Escherichia coli D21. Below pH 5, the helical content of LL-37 gradually decreases, and at pH 2 it is entirely disordered. In contrast, the helical structure is retained at pH over 13. The minimal inhibitory concentration of LL-37 against E. coli is 5 M, and at 13-25 M the peptide is cytotoxic against several eukaryotic cells. In solutions containing the ion compositions of plasma, intracellular fluid, or interstitial fluid, LL-37 is helical, and hence it could pose a danger to human cells upon release. However, in the presence of human serum, the antibacterial and the cytotoxic activities of LL-37 are inhibited.

Mechanism of Lipid Bilayer Disruption by the Human Antimicrobial Peptide, LL-37

“…The majority of the experiments were carried out using LL-37-U, synthetic LL-37 with a sequence identical to the biologically active form and no isotopic labels. CD spectroscopy was used to confirm that this synthetic peptide behaved as reported previously in the literature (18,19,25,26), exhibiting a random coil structure in pure water and a primarily helical signature in the presence of anions or lipids at micromolar peptide concentrations (data not shown). A second sample of LL-37, LL-37-L, was synthesized incorporating site-specific isotopic labels: 15 NVal 21 , 13 CdO-Leu 31 , and 13 C R -Ala 13 .…”

“…Oriented DMPC bilayers were also directly hydrated with tris buffer ( Figure 8B) to test whether the presence of ions, known to affect the structure and aggregation state of LL-37 in solution (18), also affect its interaction with lipids. For DMPC with 0% or 1% LL-37, the 31 P spectra obtained with either hydration method are identical ( Figure 8A,B) indicating that these samples are insensitive to low salt concentrations.…”

“…CD experiments have shown that LL-37 is predominantly R-helical in solution as long as anions are present (10-160 mM concentration, depending on the anion), or the concentration of peptide is above 1 µM (18). The concentration dependence and crosslinking experiments demonstrate that aggregation of LL-37 stabilizes the helical conformation (25).…”

“…Anions screen this peptide-peptide repulsion and enable LL-37 to fold into a helix and aggregate. LL-37 helicity and aggregation state in solution are positively correlated with activity (18), indicating that peptide-peptide association and high local peptide concentration are important. In lipid bilayers, similar reasoning holds that electrostatic attraction between LL-37 and negatively charged lipids should lead to regions rich in PG and peptide, and that greater disruption of the lipids should occur in anionic bilayers due to this indirect lipid-mediated peptide aggregation.…”

Effects of human cathelicidin antimicrobial peptide LL‐37 on lipopolysaccharide‐induced nitric oxide release from rat aorta in vitro

“…It was early recognized that LL-37 and LL-37 analogs have direct effects on eukaryotic cells. Johansson and colleagues (28) observed an increased fluorescein staining by fluorescein diacetate of human peripheral leukocytes and the T-cell line MOLT after incubation with LL-37 at concentrations of approximately 100 mg ml À1 . They concluded that LL-37 is toxic not only to bacteria but also to eukaryotic cells.…”