How cholesterol modulates LL-37 function: A biophysical study in eukaryotic-like membrane systems

The limited selectivity of antimicrobial peptides (AMPs) can lead to unintended damage to healthy eukaryotic cells, a process strongly influenced by membrane physicochemical properties. Cholesterol (CHO) is a key regulator of lipid packing, membrane order, and bilayer mechanical stability; however, its role in modulating AMP-membrane interactions remain incompletely understood. Here, we examine how CHO alters peptide-membrane interactions at the molecular level using eukaryotic membrane models composed of phosphatidylcholine, sphingomyelin, and increasing CHO fractions. Fourier-transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC), and fluorescence spectroscopy were employed to probe both structural and thermodynamic changes upon interaction with the human cathelicidin LL-37. FT-IR and DSC revealed that LL-37 preferentially perturbs sphingomyelin-enriched membranes, inducing shifts in phase transition temperatures and signatures of domain segregation. These perturbations were pronounced in the absence of CHO and remained detectable up to 10 mol% CHO, but were markedly attenuated at 20 mol% CHO. ITC measurements confirmed a higher binding affinity of LL-37 for sphingomyelin-rich regions, whereas fluorescence permeabilization assays demonstrated a progressive increase in membrane resistance to peptide-induced disruption with increasing CHO content. Collectively, these results suggest that LL-37 preferentially partitions into cholesterol-poor membrane domains, destabilizing their structure. In contrast, cholesterol incorporation stabilizes the surface of cholesterol-rich domain without necessarily promoting peptide anchoring within the bilayer. This study provides mechanistic insights into AMP cytotoxicity toward eukaryotic membranes and underscores cholesterol's protective role offering guidance for designing peptides with enhanced therapeutic selectivity.