Native oligomerization determines the mode of action and biological activities of human cathelicidin LL-37

LL-37 is a multifunctional component of innate immunity, with a membrane-directed antimicrobial activity and receptor-mediated pleiotropic effects on host cells. Sequence variations in its primate orthologues suggest two types of functional features have evolved; human LL-37-like peptides form amphipathic helical structures and self-assemble under physiological conditions, while rhesus RL-37-like ones only adopt this structure in the presence of bacterial membranes. The first type of peptide has a lower and more medium-sensitive antimicrobial activity than the second, but an increased capacity to stimulate host cells. Oligomerization strongly affects the mode of interaction with biological membranes and consequently both cytotoxicity and receptor-mediated activities. In this work we explored the effects of LL-37 self-association by using obligate, disulfide-linked dimers with either parallel or anti-parallel orientations. These had an increased propensity to form stacked helices in bulk-solution and when in contact with either anionic or neutral model membranes. The antimicrobial activity against Gram-positive or Gram-negative bacteria, as well as the cytotoxic effects on host cells, strongly depended on the type of dimerization. To investigate the extent of native oligomerization we replaced Phe5 with the photoactive residue p-Benzoyl-L-Phenylalanine (Bpa), which on UV irradiation enabled covalent cross-linking and allowed us to assess the extent of oligomerization in both physiological solution and in model membranes