Distinct roles of SNARE-mimicking lipopeptides during initial steps of membrane fusion

Abstract

A model system for membrane fusion, inspired by SNARE proteins and based on two complementary lipopeptides CP_nE₄ and CP_nK₄, has been recently developed. It consists of cholesterol (C), a poly(ethylene glycol) linker (P_n) and either a cationic peptide K₄ (KIAALKE)₄ or an anionic peptide E₄ (EIAALEK)₄. In this paper, fluorescence spectroscopy is used to decipher distinct but complementary roles of these lipopeptides during early stages of membrane fusion. Molecular evidence is provided that different distances of E₄ in CP_nE₄ and K₄ in CP_nK₄ from the bilayer represent an important mechanism, which enables fusion. Whereas E₄ is exposed to the bulk and solely promotes membrane binding of CP_nK₄, K₄ loops back to the lipid–water interface where it fulfills two distinct roles: it initiates bilayer contact by binding to CP_nE₄ containing bilayers; and it initiates fusion by modulating the bilayer properties. The interaction between CP_nE₄ and CP_nK₄ is severely down-regulated by binding of K₄ to the bilayer and possible only if the lipopeptides approach each other as constituents of different bilayers. When the complementary lipopeptides are localized in the same bilayer, hetero-coiling is disabled. These data provide crucial insights as to how fusion is initiated and highlight the importance of both peptides in this process.