Membranolytic Mechanism of Amphiphilic Antimicrobial β-Stranded [KL]n Peptides.

Abstract

Amphipathic peptides can act as antibiotics due to membrane permeabilization. KL peptides with the repetitive sequence [Lys-Leu]n-NH2 form amphipathic β-strands in the presence of lipid bilayers. As they are known to kill bacteria in a peculiar length-dependent manner, we suggest here several different functional models, all of which seem plausible, including a carpet mechanism, a β-barrel pore, a toroidal wormhole, and a β-helix. To resolve their genuine mechanism, the activity of KL peptides with lengths from 6–26 amino acids (plus some inverted LK analogues) was systematically tested against bacteria and erythrocytes. Vesicle leakage assays served to correlate bilayer thickness and peptide length and to examine the role of membrane curvature and putative pore diameter. KL peptides with 10–12 amino acids showed the best therapeutic potential, i.e., high antimicrobial activity and low hemolytic side effects. Mechanistically, this particular window of an optimum β-strand length around 4 nm (11 amino acids × 3.7 Å) would match the typical thickness of a lipid bilayer, implying the formation of a transmembrane pore. Solid-state 15N- and 19F-NMR structure analysis, however, showed that the KL backbone lies flat on the membrane surface under all conditions. We can thus refute any of the pore models and conclude that the KL peptides rather disrupt membranes by a carpet mechanism. The intriguing length-dependent optimum in activity can be fully explained by two counteracting effects, i.e., membrane binding versus amyloid formation. Very short KL peptides are inactive, because they are unable to bind to the lipid bilayer as flexible β-strands, whereas very long peptides are inactive due to vigorous pre-aggregation into β-sheets in solution.