Cryo-electron microscopy studies of actinoporin pores highlight the pore assembly mechanism and details of protein-membrane interactions

Actinoporins are a protein family of α-pore-forming proteins (PFPs), initially isolated from sea anemones. These proteins are key components of the venom system used by sea anemones and some other cnidarians, the most ancient extant lineage of venomous animals, where they play a role in predation and predator deterrence through rapid membrane disruption. The scientific journey to understand these potent molecules began more than five decades ago with their initial isolation from crude anemone extracts. In recent years structural studies enabled the determination of multiple structures of monomeric actinoporins in solution and in complexes with ligands, elucidating their mechanism of membrane binding and pore formation, highlighting in the fragaceatoxin C (FraC) pore structure. Recently, two groups independently produced multiple pore structures of actinoporins and a close homologue, further elucidating the mechanism of pore formation and investigating the role of lipids in the pore structure, which is even more prominent than initially thought. Hereby, we highlight the achievements of the two studies and discuss some remaining open questions. • CryoEM structures show actinoporin pores are protein–lipid assemblies, where lipids act as integral structural components. • Actinoporin pores contain ordered sphingomyelin and cholesterol lipids that stabilize pores and shape their architecture. • Membrane composition modulates pore structure and lipid occupancy. • Oligomeric intermediates provide evidence for a sequential, multistep mechanism of pore assembly. • Variable pore stoichiometries were observed and unresolved question remains how to control pore stoichiometry.