Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons1. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders2. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport)3. BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals4. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other5: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin5. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor6. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain7, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral β-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open β-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.
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We thank A. Blanc for performing the mass spectrometry analysis of the recombinant proteins and L. Knecht for help with protein production. This work was supported by UCB Pharma, UCB NewMedicines. This work was also supported by the Swiss National Science Foundation (grant 310030B_138659), The Netherlands Organization for Scientific Research (NWO-ALW-VICI) and The Netherlands Organization for Health Research and Development (ZonMW-TOP).
The authors declare no competing financial interests.
Extended data figures and tables
Side view (a) and top view (b) of SV2C-LD chain D of the complex structure. One full turn of the β-helix comprises 20 amino acids. The central hydrophobic core of the β-helix is mostly formed by stacked, slightly tilted phenylalanine residues. Cartoon representation: the helix, β-strands and loops are shown in red, yellow and green, respectively. The side chains are shown as lines in green and in atom colours. The 310-helix is indicated. The flexible N- and C-terminal regions that were not visible in the structure are schematically indicated as dotted lines.
a, Overview of the prominent interactions that were analysed by site-directed mutagenesis. The colour code in Fig. 1 is used. b, SDS–PAGE analysis of the pull-down assays. The 6×His-tagged BoNT/A domain (∼50 kDa) and the untagged SV2C domain (∼ 15 kDa) are indicated by arrows. c–f, Close-up views of specific interactions. c, The hydrogen bonds of N559. d, The hydrogen bonds of T1145/T1146. e, The cation–π stacking interaction between BoNT/A-RBD R1156 and SV2C-LD F563. f, The putative long-range electrostatic interactions of BoNT/A-RBD R1294. R1294 is not defined in the electron density of the complex structure and hence does not participate in hydrogen bonds or salt bridges. Nevertheless, mutagenesis of R1294 to alanine strongly reduces the binding of BoNT/A-RBD to SV2C-LD. We speculate that long-range electrostatic interactions between the positively charged BoNT/A-RBD arginine (depicted as a surface coloured according to the electrostatic potential) and the negatively charged regions in SV2C-LD have a role in complex formation. g, Sequences and schematic representations of the peptides that were used for the complex inhibition studies.
The affinities of wild-type and mutant SV2C-LD for BoNT/A-RBD were determined by fluorescence anisotropy titration of labelled SV2C-LD (a, c, f) and subsequent displacement with unlabelled SV2C-LD (b), SV2C-LD F563A (d) or SV2C-LD N559A (g). Alternatively, the affinities of SV2C-LD mutants were calculated from the apparent Kd of labelled SV2C-LD in the presence (green) or absence (red) of SV2C-LD F563A (22.6 μM, e) or SV2C-LD N559A (18.2 μM, h). The affinities of BoNT/A-RBD R1294A (i, j), BoNT/A-RBD R1156E (k, l) and BoNT/A-RBD T1145A/T1146A (m, n) for SV2C-LD were determined accordingly by anisotropy displacement titrations. For values, see Extended Data Table 3. Source data
Extended Data Figure 5 The BoNT/A-A2 peptide inhibits the internalization of BoNT/A-RBD by striatal neurons.
Representative images of GFP–BoNT/A-RBD uptake by cultured striatal neurons (DIV18). Neurons were pre-incubated with the GST–BoNT/A-A2 or the GST control (5 µM, 15 min) in high K+ or control buffer and treated with GFP–BoNT/A-RBD or GFP only (200 nM, 10 min) and stained for the neuronal marker tubulin-β3 (TUBB3) and endogenous VGAT to label presynaptic terminals. Scale bar, 50 µm. Representative images are from a total of 20 images per group, N = 2 independent experiments.
a, A typical example of BoNT/A-RBD (green) binding to SV2C–Flag (blue) in HEK293T cells. Cells were transfected with SV2C–Flag and mRFP (red) to highlight transfected cells, fixed and stained using Flag- and His-tagged antibodies. DAPI (4′,6-diamidino-2-phenylindole, purple) was used for visualizing all cell nuclei. Scale bar, 50 µm. b, Quantification of BoNT/A-RBD binding in SV2C–Flag expressing HEK293T cells. Cells were transfected with SV2C–Flag and mRFP, or mRFP only, and incubated with GST–BoNT/A-A2 peptide or GST control (5 µM, 15 min) and treated without (control) or with BoNT/A-RBD (100 nM, 10 min). The total area of BoNT/A-RBD was normalized to the total area of mRFP-transfected cells. No BoNT/A-RBD: 1.24 ± 0.64%, n = 8; no SV2C–Flag: 0.82 ± 0.64%, n = 5; no GST–BoNT/A-A2: 42.13 ± 7.44%, n = 9; 5 µM GST–BoNT/A-A2: 6.92 ± 1.93%, n = 9; 5 µM GST: 24.55 ± 8.01%, n = 9; Mann–Whitney U test, *** P < 0.001, * P < 0.05, NS not significant, n = number of images analysed per group, N = 2 independent experiments. All tests were performed two-sided. Data are presented as the mean ± s.e.m.
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Benoit, R., Frey, D., Hilbert, M. et al. Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A. Nature 505, 108–111 (2014). https://doi.org/10.1038/nature12732
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