Abstract
K62 is an antifungal killer toxin produced by Saccharomyces paradoxus, encoded by a double-stranded RNA satellite. The toxin exhibits a unique antifungal activity but lacks sequence homology to other killer toxins, and its antifungal mechanism of action remains unknown. To understand the function of K62, its tertiary structure was predicted using AlphaFold, followed by molecular dynamics simulations to create high-confidence molecular models. These analyses revealed that K62 monomers closely resemble the five-beta-strand domain found in pore-forming aerolysin toxins. Models of K62 oligomers yielded a circular complex and beta-barrel with structural and biochemical similarities to aerolysin-family pre-pores and pores. Consistent with the formation of aerolysin-like pores, recombinant K62 assembled into membrane-associated high molecular weight oligomers (>250 kDa) that were heat- and detergent-resistant. K62 has more than 1,000 uncharacterized sequence homologs, which were mostly found in fungi of the Ascomycota, as well as in the Chytridiomycota, Basidiomycota, plants, and bacteria, with evidence of extensive horizontal gene transfer. Homologs were also identified in pathogenic fungal species, including human and plant pathogens from the Candida and Fusarium genera, but unlike aerolysins, K62 appeared to be non-toxic to higher eukaryotes. K62 is the first aerolysin family protein discovered in yeasts, revealing a likely role in fungal niche competition and establishing an entirely new, expansive family of aerolysin-like proteins.IMPORTANCEPore-forming toxins are potent biological weapons used across nature, from virulence factors to immune defense proteins. This study identifies K62, a little-known antifungal toxin produced by a wild yeast, as a structural and functional relative of the aerolysin family, which is well-known for forming damaging pores in cell membranes. Using structure prediction, molecular simulations, and biochemical analysis, we show that K62 assembles into large, stable pore-like complexes. Remarkably, K62 is just one member of a large and previously unrecognized family of similar toxin-like proteins found in fungi, plants, and bacteria, including pathogens that affect humans and crops. These findings uncover an unexpected evolutionary link across kingdoms, suggesting that pore-forming toxins may play a widespread role in fungal pathogenesis and microbial warfare. This work lays the foundation for understanding a new group of antifungal molecules and their potential impacts on health, agriculture, and microbial ecology.