The structure and function of membrane proteins depend on their interactions with lipids that constitute membranes. Actinoporins are α-pore-forming proteins that bind preferentially to sphingomyelin-containing membranes, where they oligomerize and form transmembrane pores. Through a comprehensive cryo-electron microscopic analysis of a pore formed by an actinoporin Fav from the coral Orbicella faveolata, we show that the octameric pore interacts with 112 lipids in the upper leaflet of the membrane, reveal the roles of lipids, and demonstrate that the actinoporin surface is suited for binding multiple receptor sphingomyelin molecules. When cholesterol is present in the membrane, it forms a cluster of four molecules associated with each protomer. Atomistic simulations support the structural data and reveal additional effects of the pore on the lipid membrane. These data reveal a complex network of protein-lipid and lipid-lipid interactions and an underrated role of lipids in the structure and function of transmembrane protein complexes.

Pore-forming proteins perforate lipid membranes and consequently affect their integrity and cell fitness. Therefore, it is not surprising that many of these proteins from bacteria, fungi, or certain animals act as toxins. While pore-forming proteins have also been found in plants, there is little information about their molecular structure and mode of action. Bryoporin is a protein from the moss Physcomitrium patens, and its corresponding gene was found to be upregulated by various abiotic stresses, especially dehydration, as well as upon fungal infection. Based on the amino acid sequence, it was suggested that bryoporin was related to the actinoporin family of pore-forming proteins, originally discovered in sea anemones. Here, we provide the first detailed structural and functional analysis of this plant cytolysin. The crystal structure of monomeric bryoporin is highly similar to those of actinoporins. Our cryo-EM analysis of its pores showed an actinoporin-like octameric structure, thereby revealing a close kinship of proteins from evolutionarily distant organisms. This was further confirmed by our observation of bryoporin’s preferential binding to and formation of pores in membranes containing animal sphingolipids, such as sphingomyelin and ceramide phosphoethanolamine; however, its binding affinity was weaker than that of actinoporin equinatoxin II. We determined bryoporin did not bind to major sphingolipids found in fungi or plants, and its membrane-binding and pore-forming activity was enhanced by various sterols. Our results suggest that bryoporin could represent a part of the moss defense arsenal, acting as a pore-forming toxin against membranes of potential animal pathogens, parasites, or predators.

The bacterium Bacillus thuringiensis (Bt) produces insecticidal proteins during the sporulation phase. These proteins are located in parasporal crystals consisting of two delta-endotoxin classes, crystal (Cry) and cytolytic (Cyt) toxins. In vitro, Cyt toxins show cytolytic activity against bacterial and a variety of insect and mammalian cells. They bind to cell membranes with unsaturated phospholipids and sphingomyelin. Although Bt and its parasporal crystals containing both Cry and Cyt toxins have been successfully used as bioinsecticides, the molecular mechanism of action of Cyt toxins is not yet fully understood. To address this, we exposed Cyt2Aa to lipid membranes and visualized membrane disruption process using cryo-electron microscopy. We observed two types of Cyt2Aa oligomers. First, Cyt2Aa forms smaller curved oligomers on the membrane surface that become linear over time, and detach when the membrane ruptures. Similar linear filamentous oligomers were also formed by Cyt2Aa in the presence of detergents without prior exposure to lipid membranes, which exhibited attenuated cytolytic activity. Furthermore, our data suggest that Cyt2Aa adopts different conformations between its monomeric and oligomeric forms. Overall, our results provide new evidence for a detergent-like mechanism of action of Cyt2Aa rather than the pore-forming model of target membrane disruption of this important class of insecticidal proteins.

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4^E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry.

Cone snails are a large group of marine gastropods that produce a complex mixture of toxic compounds to hunt prey and defend against predators. The majority of the venom comprises small toxic peptides named conotoxins, which target membrane receptors. In contrast, a smaller part of the venom contains larger proteins and conoproteins, which are thought to be involved in conotoxin maturation and the envenomation process, respectively. Interestingly, many species of cone snails contain conoporins, which are similar to actinoporins—pore-forming toxins found in sea anemones. These actinoporin-like proteins (ALPs) have recently been detected in many molluscan species, and only a few have been experimentally characterized. Due to being highly expressed in the venom gland of many cone snail species, conoporins are thought to play an important part in the envenomation process. Despite this, the exact function of conoporins is currently unknown. We propose several hypotheses aiming to elucidate their biological role.

The use of protein nanopores has proven to be very promising for the identification of proteins or the sequencing of peptides. However, their widespread use in biosensor applications is limited by more complex molecular properties of proteins in comparison to nucleic acids. Here, we developed an α-helical Fav nanopore from the coral Orbicella faveolata to detect human histones using a commercial MinION device. The engineered nanopores showed stable insertion into the polymeric membrane support. Bulk analysis of several tens to hundreds of pores per measurement revealed significant differences in mean current passing the pore and current noise resulting from capturing different full-length human histones or their post-translationally modified variants into the pore lumen. Importantly, detailed blocking analyses confirmed histone-specific signals that allow further discrimination between two medically important extracellular histones in mixtures. The newly developed Fav nanopore and high-throughput approach for the detection of biomedically important proteins is an important step towards the widespread use of nanopores in modern analytics.