How ostreolysin A detects SM/cholesterol in membranes.

Abstract

Some pore-forming toxins target cells by binding specific lipids on the plasma membrane. Structural investigation of these protein-lipid interactions can provide insight into how membrane lipids are presented to proteins on the extracellular surface of cells. We use toxin proteins that recognize distinct regulatory pools of cholesterol in mammalian plasma membranes to better understand cholesterols membrane organization. The fungal toxin Ostreolysin A (OlyA) specifically binds sphingomyelin (SM) only when SM is complexed with cholesterol in the SM-sequestered pool. We’ve rigorously tested the lipid specificity of OlyA in reconstituted and cellular systems and discovered a single mutation in OlyA that abolishes SM/cholesterol specificity. This mutant, OlyA (E69A), binds SM containing membranes regardless of cholesterols presence. High-resolution X-ray structures of WT and mutant OlyA have led to a working model proposing these proteins recognize different membrane conformations of SM. Unfortunately, our solution structures lack direct information about sphingomyelins conformation in a membrane environment. Using single-particle Cryo-EM, we’ve now solved structures of OlyA bound to SM containing membranes with and without cholesterol. These structures provide the first direct insights into how WT and mutant OlyA recognize SM in these two forms. They also provide high-resolution information about the protein-protein interactions that drive pore assembly and shed light on how protein-lipid interactions initiate this process.