Physicochemical Membrane Properties Reveal a Structural Element Involved in the Adaptation of Actinoporins to Cholesterol-Rich Membranes

Abstract

The plasma membrane houses a complex mixture of membrane proteins and lipids, and is also the specific target of amphitropic proteins such as pore-forming toxins. The activity of all of these proteins is exquisitely regulated by the physicochemical landscape of the lipid bilayer. Actinoporins, cytolysins produced by sea anemones, are toxins that form transmembrane pores in membranes showing lipid phase separation, especially those containing sphingomyelin. Fragaceatoxin C (FraC), an actinoporin from Actinia fragacea, is activated by lipid-phase separation, but a description of the molecular basis behind the adaptation of the protein to membranes with different physicochemical properties and lipid composition is still obscure. In this work, we show that FraC contains a key conserved residue (Phe16) involved in cholesterol sensing. Mutations on Phe16 generated protein species that were not active in cholesterol-rich membranes regardless of the nature of the residue used in the substitution. In contrast, the lytic activity of wild-type FraC and the muteins was essentially identical. A detailed analysis shows that the lytic activity of the Phe16-defective toxin measured in raft-like model membranes was inversely correlated with the concentration of cholesterol in the membrane. This behavior can be explained by the segregation of liquid-ordered domains (cholesterol-rich) at the expense of liquid-disordered domains (rich in unsaturated phosphatidylcholines). These results describe complementary mechanisms of membrane recognition that have evolved in response to different membrane environments.