In vitro evolution driven by epistasis reveals alternative cholesterol-specific binding motifs of perfringolysin O.

The crucial molecular factors that shape the interfaces of lipid-binding proteins with their target ligands and surfaces remain unknown due to the complex makeup of biological membranes. Cholesterol, the major modulator of bilayer structure in mammalian cell membranes, is recognised by various proteins, including the well-studied cholesterol-dependent cytolysins (CDCs). Here, we use in vitro evolution to investigate the molecular adaptations that preserve the cholesterol specificity of perfringolysin O (PFO), the prototypical CDC from Clostridium perfringens. We identify variants with altered membrane-binding interfaces whose cholesterol-specific activity exceeds that of the wild-type PFO. These novel variants represent alternative evolutionary outcomes and have mutations at conserved positions that can only accumulate when epistatic constraints are alleviated. Our results improve the current understanding of the biochemical malleability of the surface of a lipid-binding protein.

Sequestration of membrane cholesterol by cholesterol-binding proteins inhibits SARS-CoV-2 entry into Vero E6 cells.

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 D4E458L 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.

Littoporins: Novel actinoporin-like proteins in caenogastropod genus Littorina.

In the course of searching for genes controlling the immune system in caenogastropod mollusks, we characterized and phylogenetically placed five new actinoporin-like cytolysins expressed in periwinkles of the genus Littorina. These newly discovered proteins, named littoporins (LitP), contain a central cytolysin/lectin domain and exhibit a predicted protein fold that is almost identical to the three-dimensional structures of actinoporins. Two of these proteins, LitP-1 and LitP-2, were found to be upregulated in L. littorea kidney tissues and immune cells in response to natural and experimental infection with the trematode Himasthla elongata, suggesting their potential role as perforins in the systemic anti-trematode immune response. The primary sequence divergence of littoporins is hypothesized to be attributed to the taxonomic range of cell membranes they can recognize and permeabilize.