Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A

•Neutralising mechanism of potent therapeutic monoclonal BoNT/A antibody is exhibited.•Single mutation G1292R eliminates interaction of BoNT/A with all three SV2 isoforms.•Proof for neuronal uptake of BoNT/A by a double receptor mechanism.•Inactive quintuple BoNT/A mutant represents ideal antigen for vaccination.

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (HC). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the HCC and HCN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties.

Structured summary of protein interactionsHCAbinds to rSV2A by pull down (View interaction)BoNT/Abinds to rSV2C by pull down (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)HCAphysically interacts with SV2A and SV2B by anti tag coimmunoprecipitation (View interaction)HCEphysically interacts with SV2A and SV2B by anti tag coimmunoprecipitation (View interaction)HCFphysically interacts with SV2A and SV2B by anti tag coimmunoprecipitation (View interaction)