Molecular architecture of black widow spider neurotoxins

Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (-latrotoxin (-LTX)), five specialized on insects (, {beta}, {gamma}, {delta}, {varepsilon}-latroinsectotoxins (LITs), and one on crustaceans (-latrocrustatoxin (-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the -LCT monomer and the {delta}-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel -helices. Both domains are flexibly linked via an N-terminal -helical domain and a small {beta}-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant -LCT and {delta}-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between -LCT and {delta}-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.