Pore-forming proteins (PFPs) compromise membrane integrity by forming pores. These proteins are typically secreted as soluble monomers that bind to membranes, oligomerize and undergo conformational changes to form transmembrane pores. Actinoporins, a family of α-PFPs found in sea anemones, specifically target membranes containing sphingomyelin (SM). The presence of cholesterol in the membrane further enhances the activity of actinoporins. The first detailed structure of an actinoporin pore was reported for fragaceatoxin C from the strawberry anemone (Actinia fragacea).
We investigated the pore structure of Fav, an actinoporin from the mountainous star coral (Orbicella faveolata), using cryo-electron microscopy (cryo-EM). Fav pores were prepared on lipid vesicles with varied lipid compositions to study their structure. For high-resolution structural analysis, pores were extracted from vesicles using detergents. The resulting cryo-EM maps revealed a complex network of lipids associated with the Fav pore. In membranes containing cholesterol, a higher number of lipids, including densely packed cholesterol molecules beneath the pore cap, were observed.
Phospholipids associated with the pore were categorized into three distinct groups based on their roles and positions. Receptor lipids, located in the membrane-binding region, facilitate initial protein binding. Structural lipids between two protomers interact with both and stabilize the complex. Bridging lipids, found at the pore’s outer edge, with limited protein interactions but stabilize the structure by linking adjacent protomers.
Molecular dynamics simulations of the pore in the membrane confirm the stable binding of observed lipids and reveal that the pore affects the mobility of membrane lipids in its surroundings not in direct contact with the protein. This effect is less pronounced in the lower leaflet of the membrane where no lipids are specifically bound by the pore. Our findings highlight the critical and diverse roles of lipids in Fav pore formation and stability.