Fig 2: An engineered SV2A–SV2C chimera maintains an SV2A-like binding to BoNT/E.a A schematic diagram showing the designs of SV2A–SV2C chimeras where the sequences of SV2C-L4 and SV2A-L4 are shown as blue and red hollow bars, respectively. The structure of SV2C-L4 is shown as a blue cartoon with residues K518, I538, N559, and the N559 glycan shown as sticks (PDB: 5JLV. b The chimeric SV2Ac–G6AA fusion protein is mono-dispersed in solution based on a gel-filtration analysis. c A pull-down assay was performed using HCE as prey and the His-tagged SV2-L4 or SV2–G6AA fusion proteins as baits. Only SV2A–G6AA and SV2Ac–G6AA were able to pull down HCE (indicated by asterisks), but not other SV2–G6AA fusion proteins or the stand-alone SV2-L4 variants. A representative result is shown (n = 3). d and e The full-length SV2A and SV2Ac were expressed via lentiviral transduction in mouse cortical neurons cultured from SV2A/B double knockout (KO) mice. Neurons were exposed to HCE (5 min) (d) or BoNT/E at the indicated concentration (24 h in medium) (e). Cell lysates were harvested and analyzed by immunoblot assays. The synaptic vesicle protein, Synaptotagmin 1 (Syt-1), was detected as a loading control. The SNAP-25 antibody can detect both the full-length SNAP-25 and the fragment generated after cleavage by BoNT/E (marked with *). SV2A(+/+)/SV2B(-/-) neurons were analyzed in parallel as a positive control. A representative result is shown (n = 2). Source data are provided as a Source Data file.

Fig 3: Adaptive changes of TH-positive interneurons and SV2C-positive cholinergic interneurons in Aux-KO/SJ1-KIRQ striatum.a Immunostaining of TH reveals the Aux-KO/SJ1-KIRQ striatum contains a large number of THINs. THINs are not found in Ctrl and Aux-KO striatum; and are less often found in SJ1-KIRQ striatum. Scale bar: 20 µm. b Quantification for the number of THINs in 10 randomly selected regions of interest (ROIs) from a representative coronal section of the striatum. Data are represented as mean ± SEM (**p < 0.01 by one-way ANOVA with post-hoc Tukey’s test). n = 3–4 mice for each genotype. c Immunostaining of TH (green) and DAT (red) reveals presence of TH-positive/DAT-negative neurons in the 1-month-old Aux-KO/SJ1-KIRQ striatum. Scale bar: 20 µm. d THINs also show immunoreactivity towards anti-AADC antibody. Scale bar: 20 µm. e Representative images of SV2C and TH staining in the dorsal striatum of 1-month-old Aux-KO/SJ1-KIRQ mice. TH and SV2C were observed to be expressed in separate neuron populations. SV2C-positive/TH-negative neurons are marked by white arrows. Scale bar: 20 µm. f Double staining of striatal region with cholinergic marker ChAT and SV2C shows overlap between the two in a specific neuronal subset, indicating that the SV2C-positive neurons are giant striatal ChINs. Scale bar: 20 µm. g The cell bodies of THINs and ChINs are selected as our region of interest to perform Pearson’s correlation coefficient. This demonstrate the two different types of interneurons: THINs where TH and AADC colocalizes well but not with DAT; ChINs where SV2C colocalizes well with ChAT but not with TH. Data are represented as mean ± SEM. n = 7–10 random sampling site. h Striatal IHC for single coronal section reveals subset of ChAT-positive ChINs that exhibit striking upregulation of SV2C in Aux-KO/SJ1-KIRQ mice compared to control. Scale bar: 50 µm. i Quantification for percentage of ChAT-positive interneurons expressing SV2C, as well as individual intensity of SV2C and ChAT in striatal ChINs. For each individual mouse, 20 ChINs across a single coronal section of striatum were randomly selected for quantification. Data are represented as mean ± SEM (*p < 0.05 and **p < 0.01, by Student’s unpaired t test). Ctrl: n = 5 mice and Aux-KO/SJ1-KIRQ: n = 5 mice.

Fig 4: Proposed models for simultaneous binding of BoNT/E and BoNT/A to the membrane-anchored SV2 and gangliosides.a Proposed binding modes of BoNT/E (PDB: 3FFZ) and BoNT/A (PDB: 3BTA) with the membrane-bound SV2 and gangliosides. The holotoxins are positioned based on the structures of the HCE–SV2Ac and the HCA–SV2C (PDB: 5JLV) complexes. A representative complex-type N-glycan (red sticks, PDB: 3QUM) is modeled to represent the N-glycan of SV2A. The gangliosides GD1a and GT1b are modeled based on the structures of HCE–GD1a (PDB: 7OVW) and HCA–GT1b (PDB: 2VU9). b The structures of the HCE–SV2Ac and the HCA–SV2C (PDB: 5JLV) complexes with the modeled gangliosides are superimposed based on HCE and HCA. The view angle on the left panel is identical to that shown in panel (a). A different view with a rotation of ~115° about a vertical axis is shown on the right panel. Two equivalent ß-sheets (ß8) on SV2Ac-L4 and SV2C-L4 are highlighted as a marker to show the similar orientations of SV2Ac and SV2C relative to the membrane in this putative model. c Surface representation of BoNT/E holotoxin in complex with SV2A (orange) and GD1a (cyan sticks). BoNT/E adopts a “closed-wing” conformation, in which HCE (lemon) and LC/E (pale cyan) are located on the same side of the long helical HNE (purple). d Surface representation of BoNT/A holotoxin in complex with SV2C (blue) and GT1b (deep teal sticks). BoNT/A has an “open-wing” conformation, in which HCA (light purple) and LC/A (yellow) are located on the opposite sides of the long helical HNA (brown).

Fig 5: Structure of the sialic acid-bound HCE–SV2Ac complex and comparison of the glycan-binding modes between BoNT/E and BoNT/A.a Cartoon representation of the sialic acid (SIA)-bound HCE–SV2Ac complex with HCE colored in lemon, SV2Ac in orange, and sialic acid in red. An omit electron density map for sialic acid contoured at 1.5s was overlaid with the final refined model. b A close-up view of the interactions between HCE and sialic acid. One water molecule (W) that mediates the HCE–SIA binding is shown as a cyan sphere. c The HCE–SV2Ac–SIA complex and the HCA–SV2C complex (PDB: 5JLV) were superimposed based on HCE (lemon) and HCA (blue-white). The SIA (red), NAG (orange), and SV2C glycan (blue) are shown as sticks. The SIA-binding and the SV2C-glycan-binding pockets are highlighted in red and blue boxes, respectively. d A close-up view into the SV2C-glycan-binding pocket reveals the differences between HCA (blue-white) and HCE (lemon) in this area. HCE residue Q1250 would clash with the SV2C glycan based on the superposition. e A close-up view into the SIA-binding pocket shows that this pocket is partly conserved between HCE and HCA.