Concentration-Dependent, Membrane-Selective Activity of Human LL37 Peptides Modified with Collagen Binding Domain...

Antimicrobial peptides (AMPs) such as LL37 are promising alternatives to antibiotics to treat wound infections due to their broad activity, immunomodulatory functions, and low likelihood of antimicrobial resistance. To deliver LL37 to chronic wounds, we developed two chimeric LL37 peptides with C-terminal collagen binding domains (CBD) derived from collagenase ( cCBD-LL37) and fibronectin ( fCBD-LL37) as a strategy for noncovalent tethering of LL37 onto collagen-based, commercially available wound dressings. The addition of CBD sequences to LL37 resulted in differences in cytotoxicity against human fibroblasts and antimicrobial activity against common wound pathogens. In this study, we sought to determine the sequence-, structure-, and concentration-dependent properties underlying these differences in bioactivity. Molecular dynamics (MD) simulations allowed visualization of the structure of each peptide and calculation of residue-level helicity, revealing that residues within the CBD domains were not helical. Circular dichroism (CD) spectroscopy affirmed that the overall structures of LL37 and each CBD-LL37 peptide was primarily helical (greater than 67%) in a membrane-like solvent. Quartz crystal microbalance with dissipation (QCM-D) and imaging of fluorescent bilayers revealed unique, concentration-dependent interactions of each peptide with bilayers of different lipid compositions. Specifically, fCBD-LL37, which is less cytotoxic than LL37 and cCBD-LL37, demonstrated higher affinity toward anionic bilayers (model bacterial cell membranes) than zwitterionic bilayers (model mammalian cell membranes). In contrast, cCBD-LL37 and LL37 demonstrated similar affinities to both types of bilayers. This study demonstrates that the combination of MD, CD, and QCM-D may enable predictive modeling of the effects of primary sequence alterations on peptide secondary structure and membrane interactions. Understanding the structural and mechanistic properties of AMPs and their interactions with specific lipid bilayer compositions may enable the engineering of less cytotoxic AMPs with improved therapeutic indexes for human wound healing applications.