Gangliosides interact with synaptotagmin to form the high-affinity receptor complex for botulinum neurotoxin B

Abstract

Botulinum neurotoxin type B (BoNT/B) recognizes nerve terminals by binding to 2 receptor components: a polysialoganglioside, predominantly GT1b, and synaptotagmin 1/2. It is widely thought that BoNT/B initially binds to GT1b then diffuses in the plane of the membrane to interact with synaptotagmin. We have addressed the hypothesis that a GT1b–synaptotagmin cis complex forms the BoNT/B receptor. We identified a consensus glycosphingolipid-binding motif in the extracellular juxtamembrane domain of synaptotagmins 1/2 and confirmed by Langmuir monolayer, surface plasmon resonance, and circular dichroism that GT1b interacts with synaptotagmin peptides containing this sequence, inducing α-helical structure. Molecular modeling and tryptophan fluorescence spectroscopy were consistent with the intertwining of GT1b and synaptotagmin, involving cis interactions between the oligosaccharide and ceramide moieties of GT1b and the juxtamembrane and transmembrane domains of synaptotagmin, respectively. Furthermore, a point mutation on synaptotagmin, located outside of the BoNT/B-binding segment, inhibited GT1b binding and blocked GT1b-induced potentiation of BoNT/B binding to synaptotagmin-expressing cells. Our findings are consistent with a model in which a preassembled GT1b–synaptotagmin complex constitutes the high-affinity BoNT/B receptor.

Structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against SARS-CoV-2 infection

“…Both features have been shown to be key determi-nants of the recognition of sialic acids and gangliosides by proteins [20 , 26] . Modelling approaches have been used successfully to decipher various molecular mechanisms of protein-sugar interactions accounting for the interaction of virus [27] , bacteria [28] , membrane [13] and amyloid proteins [20] with cell surface glycolipids. This in-silico strategy was applied to unravel the molecular mechanisms underlying the antiviral mechanisms of CLQ and CLQ-OH against SARS-CoV-2 infection.…”

“…Docked complexes were subsequently submitted to iterative cycles of molecular dynamics using the CHARMM36 force field optimized for carbohydrates [17] . Interaction energies were calculated from stable complexes using the Ligand Energy Inspector function of Molegro [13] .…”

Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins

“…As expected, JLI-G10 did not disturb H C B binding to the soluble GST-Syt II ( Figures 2D and S7E). However, JLI-G10 completely blocked the binding of H C B to GST-Syt II (1-90) embedded in Triton X-100 micelles, even in the presence of co-receptor GT1b (Figures 2E and S7F) that should form a cis-complex with Syt II in the detergent micelle to enhance H C B binding (Flores et al, 2019). These data collectively suggest that JLI-G10 inhibits the binding of BoNT/B1 to neuronal membrane by simultaneously occupying the membrane-binding H C -loop and the ganglioside-binding pocket on H C B.…”

Neuronal selectivity of botulinum neurotoxins

“…GT1b (Flores et al, 2019). Not only the distribution of Syt, but also the appropriate conformation of both Syt and gangliosides seem determinant for the selective BoNT binding to neuronal cells, and probably to specific membrane areas of neuronal cells.…”