Molecular architecture of black widow spider neurotoxins

Abstract

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 (α-latrotoxin (α-LTX)), five specialized on insects (α, β, γ, δ, ε- latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-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 α-LCT monomer and the δ-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 α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small β-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 α-LCT and δ-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 α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.

Molecular mechanism of α-latrotoxin action

“…The efficiency of this process is significantly increased in the presence of receptors 20 . Recently, we reported cryo-EM structures of both the α-LCT monomer and the δ-LIT dimer, shedding light on the overall domain organization of the LTX family 19 . However, the mechanism of LTX pore formation remained elusive, as we currently lack detailed structures of a LTX tetramers and pore events have only been visualized at low resolution 18 .…”

Bacterial pore-forming toxins

“…Pore-forming toxins are widespread in both Gram-positive and Gram-negative bacteria. They are additionally produced by higher organisms, such as Anemonae and Cnidaria [11,200], indicating that they evolved quite early and represent an ancient attack form that, for bacteria, can be employed for both inter-bacterial competition and pathogenesis. While advances in the characterisation of PFTs are remarkable, many challenges and new questions remain open.…”

CryoSTAR: Leveraging Structural Prior and Constraints for Cryo-EM Heterogeneous Reconstruction

“…Finally, we demonstrate the ability of cryoSTAR to resolve the continuous heterogeneity on a small protein. α-latrocrustatoxin (α-LCT) is one of the seven black widow spider neurotoxins with a molecular weight of 130 kDa 26 . A recent work found two different conformations of α-LCT via discrete 3D classification, where the curvature of the Ankyrin-like Repeat Domain (ARD) alters, so that the tail of ARD can move towards the head domain.…”

“…Without excluding the lipid nanodisc region by manual masking or particle subtraction, cryoSTAR successfully resolves its conformational heterogeneity. Finally, we show the performance of cryoSTAR on α-latrocrustatoxin (α-LCT) 26 (EMPIAR-10827), a small protein with a molecular weight of 130 kDa. We resolve the continuous motion that agrees with the original paper found by discrete 3D classification 26 , and also uncover a different type of flexible motion.…”

“…Finally, we show the performance of cryoSTAR on α-latrocrustatoxin (α-LCT) 26 (EMPIAR-10827), a small protein with a molecular weight of 130 kDa. We resolve the continuous motion that agrees with the original paper found by discrete 3D classification 26 , and also uncover a different type of flexible motion. Structural regularization is especially beneficial in challenging cases, such as resolving continuous motions in membrane proteins or in smaller proteins.…”