Domain loss enabled evolution of novel functions in the snake three-finger toxin gene superfamily

Three-finger toxins ( 3FTXs ) are a functionally diverse family of toxins, apparently unique to venoms of caenophidian snakes. Although the ancestral function of 3FTXs is antagonism of nicotinic acetylcholine receptors, redundancy conferred by the accumulation of duplicate genes has facilitated extensive neofunctionalization, such that derived members of the family interact with a range of targets. 3FTXs are members of the LY6/UPAR family, but their non-toxin ancestor remains unknown. Combining traditional phylogenetic approaches, manual synteny analysis, and machine learning techniques (including AlphaFold2 and ProtT5 ), we have reconstructed a detailed evolutionary history of 3FTXs. We identify their immediate ancestor as a non-secretory LY6, unique to squamate reptiles, and propose that changes in molecular ecology resulting from loss of a membrane-anchoring domain and changes in gene expression, paved the way for the evolution of one of the most important families of snake toxins. 3-finger toxins are unique to the venoms of caenophidian snakes. This study traces the evolution of these toxins in snakes, highlighting a key shift from membrane-bound to secretory proteins. This transformation, involving the loss of a membrane-anchoring domain and changes in gene expression, paved the way for their venomous function.