Visualization of the interaction between sphingomyelin and cholesterol in lipid bilayer membranes

Biomembranes are complex two-dimensional liquids composed of hundreds of lipid species that interact in a myriad of ways. One such interaction, that between sphingomyelin (SM) and cholesterol in plasma membranes of animal cells, provides many functional benefits, including protection from microbial infection, prevention of unrestrained cell growth, and proper maintenance of cellular lipid composition. Owing to the liquid nature of membranes, the structure of the SM/cholesterol interaction, or any other functionally critical lipid-lipid interaction, has remained elusive. Here, we overcome this challenge using a fungal toxin called Ostreolysin A (OlyA), that has been shown to specifically bind to SM/cholesterol complexes in membranes. We used OlyA to stabilize the SM/cholesterol interaction much in the same way as antibodies are used to stabilize preexisting protein complexes. Cryoelectron microscopy analysis of OlyA bound to SM/cholesterol membranes reveals the details of the tight interaction between these two lipids-the steroid nucleus of cholesterol packs against the acyl chains of SM, and a hydrogen bond forms between the nitrogen on SM's ceramide base and the oxygen on cholesterol's hydroxyl group, thus sequestering this key functional group of cholesterol. The importance of hydrogen bonding in stabilizing the SM/cholesterol interaction is supported by structural analysis of a mutant form of OlyA that binds free SM in a cholesterol-independent manner. These results provide structural insights into the organization of cholesterol in membranes.