Structural venomics: evolution of a complex chemical arsenal by massive duplication and neofunctionalization of a single ancestral fold

Spiders are the most successful venomous animals on the planet, with more than 47,000 extant species. Most spider venoms are dominated by disulfide-rich peptides (DRPs) with a diverse range of pharmacological activities. Although some venoms contain thousands of unique peptides, little is known about the mechanisms used to generate such complex chemical arsenals. We used a combined transcriptomic, proteomic and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins, more than described for any other arachnid. We show that 15 of the 26 DRP superfamilies form an ultra-stable inhibitor cystine knot motif, and that these DRPs are the major contributor to the diversity of the venom peptidome. NMR data reveal that most of these DRPs are structurally related and range in complexity from simple to highly elaborated knottin domains that likely evolved from a single ancestral fold.