How the widely used botulinum neurotoxin A (BoNT/A) recognizes and enters neurons is poorly understood. We found that BoNT/A enters neurons by binding to the synaptic vesicle protein SV2 (isoforms A, B, and C). Fragments of SV2 that harbor the toxin interaction domain inhibited BoNT/A from binding to neurons. BoNT/A binding to SV2A and SV2B knockout hippocampal neurons was abolished and was restored by expressing SV2A, SV2B, or SV2C. Reduction of SV2 expression in PC12 and Neuro-2a cells also inhibited entry of BoNT/A, which could be restored by expressing SV2 isoforms. Finally, mice that lacked an SV2 isoform (SV2B) displayed reduced sensitivity to BoNT/A. Thus, SV2 acts as the protein receptor for BoNT/A.
De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37–43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing.
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