Fig 1.
Characterizing binding of HC/B, /C, /D, /DC, and /G to lipid membranes in nanodiscs.
(A) Structural overlay (top panel) and protein sequences (lower table) of the LBLs of HC/B, /C, /D, /DC, and /G. (B) Schematic drawing of nanodiscs (ND1). (C) The presence of nanodiscs was confirmed using negative staining EM. Scale bar represents 25 nm. (D–H). Binding of HC/B (panel D), HC/C (panel E), HC/D (panel F), HC/DC (panel G), and HC/G (panel H) to ND1 was examined using BLI assay. SA biosensors were exposed to three different concentrations of HCs (10, 25, and 50 μM) for 40 seconds (association phase), followed by an 80-second washing step (dissociation phase). Representative sensorgrams were color-coded (green: 10 μM; pink: 25 μM; blue: 50 μM) and shown, with the y-axis representing the shift in light wavelength upon binding (response nm). HC/B and HC/D did not show any detectable binding to ND1. HC/DC showed the strongest binding to ND1, and HC/C showed a modest level of binding, whereas HC/G showed low levels of binding. (I) The maximal binding signals of HC/B, /C, /D, /DC, and /G to ND1 as described in panels D–H were averaged and plotted (mean ± SD, n = 3). HC/DC showed the highest binding to ND1 among all HCs. Numerical values for (I) are available in S1 Data. BLI, biolayer interferometry; BSA, bovine serum albumin; EM, electron microscopy; HC, C-terminal receptor-binding domain; LBL, lipid-binding loop; ND1, receptor-free nanodisc; SA, streptavidin.
Fig 2.
Mutating aromatic residues at the tip of HC/DC-LBL reduces its binding to lipid membranes.
(A) Structure of the HC/DC-LBL showing the three aromatic residues at the tip. (B–D) Binding of HC/DC-LBL containing the indicated point mutations to ND1 was analyzed by BLI assays. Mutating the three aromatic residues at the tip of LBL reduced binding of HC/DC to ND1. (E) The maximal binding signals of HC/DC mutants as measured in panels B–D were averaged and plotted (mean ± SD, n = 3). Numerical values for (E) are available in S1 Data. (F) HC/DC-LBL (F1253W) showed robust binding to ND1. BLI, biolayer interferometry; BSA, bovine serum albumin; HC, C-terminal receptor-binding domain; LBL, lipid-binding loop; ND1, receptor-free nanodisc; WT, wild-type.
Table 1.
Kinectics and affinity analysis of HC/DC and the indicated mutants to ND-Syt or free Syt II (1–61).
Fig 3.
Introducing aromatic residues at the tip of HCB-LBL enables its binding to lipid membranes.
(A) Structure of HCB-LBL, with three residues at the tip marked. (B–C) Binding of HC/B mutants containing either a single point mutation, I1248W, or double point mutations, I1248W/V1249W, to ND1 were analyzed by BLI assays. HC/BW showed low-level binding to ND1, whereas HC/BWW showed robust binding. (D) The maximal binding signals of HC/B mutants as measured in panels B and C were averaged and plotted (mean ± SD, n = 3). Numerical values for (D) are available in S1 Data. (E–F) Binding of WT HC/B, HC/BW, and HC/BWW to liposomes containing POPC or POPC plus gangliosides were analyzed by liposome flotation assays. Proteins bound to liposomes floated to the top of the sucrose density gradient and were collected and subjected to immunoblot analysis. HC/BW and HC/BWW were able to bind POPC liposomes, and the presence of gangliosides further enhanced their binding. BLI, biolayer interferometry; BoNT, botulinum neurotoxin; BSA, bovine serum albumin; HC, C-terminal receptor-binding domain; LBL, lipid-binding loop; ND1, receptor-free nanodisc; PC, phosphatidylcholine; POPC, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; WT, wild-type.
Fig 4.
Lipid-binding capability synergizes with BoNT/B-CGs interactions.
(A–C) Binding of WT HC/B and HC/BWW to ND-CGs was analyzed by BLI assays. Binding kinetics were analyzed and listed in S1 Table. (D–F) Binding of WT HC/B and HC/BWW to ND-Syt was analyzed by BLI assays using the Octet RED 384 system at the indicated concentrations. Binding curves were fitted using a 1:1 binding model, and fitted lines are shown in red. Binding kinetics were analyzed and listed in S1 Table. (G–I) Binding of WT HC/B and HC/BWW to ND-Syt-CGs, which contains both Syt II and CGs, was analyzed by BLI assays using the Octet RED 384 system at the indicated concentrations. Interactions involve at least three heterogeneous components, and it is not possible to draw a precise binding KD for comparison. Binding curves were fitted using a 1:1 binding model, and fitted lines are shown in red. Estimated KD using a single–binding site model is included in S1 Table. BLI, biolayer interferometry; BoNT, botulinum neurotoxin; BSA, bovine serum albumin; CG, complex ganglioside; HC, C-terminal receptor-binding domain; KD, equilibrium dissociation constant; ND, nanodisc; Syt, synaptotagmin; WT, wild-type.
Fig 5.
Characterizing binding of HC/BWW on cultured neurons and BoNT/BMY-WW in vivo.
(A) Binding of WT HC/B, HC/BW, and HC/BWW to cultured rat cortical neurons was examined by immunoblot analysis. Neurons were exposed to HA-tagged HC/B, HC/BW, and HC/BWW for 5 minutes, washed, and harvested for immunoblot analysis. Actin served as an internal loading control. The total represents 50 ng of HC proteins. Representative blots and the intensity relative to total HC/B quantification results are shown. (B) Binding of HC/B and HC/BWW to cultured mouse cortical WT and GD2 KO neurons was examined by immunostaining analysis. Neurons were exposed to HA-tagged HC/B, HC/BWW, or HC/BWW plus GST-tagged recombinant Syt II (1–61, 10-fold of HC/BWW concentration) for 5 minutes, washed, fixed, and subjected to immunostaining analysis. Synapsin serves as an internal marker for presynaptic terminals. HC/BWW showed enhanced binding to neurons compared with HC/B and colocalized with Synapsin. Binding of HC/BWW is lower on GD2 KO neurons compared with WT neurons, and residual binding of HC/BWW on GD2 KO neurons is eliminated by recombinant Syt II fragment protein as a receptor decoy. Scale bar represents 30 μm. (C) The control BoNT/BMY elicited a dose-dependent response in the DAS mean score. Data are means ± SEM of n = 7 or 8. The response within the first 4 days is shown in the enlarged insert. (D) BoNT/BMY-WW showed a more potent response and longer duration in the DAS assay than the control BoNT/B (panel C). Data are means ± SEM of n = 7 or 8. Numerical values for (A), (C), and (D) are available in S1 Data. BoNT, botulinum neurotoxin; DAS, Digit Abduction Score; GD2 KO, Galgt1 knockout; GST, glutathione S-transferase; HA, human influenza hemagglutinin; HC, C-terminal receptor-binding domain; Syt, synaptotagmin; WT, wild-type.
Table 2.
Mean ED50, 0%BW, and LD50 following IM injection in mice.
Fig 6.
Cocrystal structure of HC/BWW in complex with Syt I peptide and ganglioside oligosaccharide.
(A) Cocrystal structure of HC/BWW (blue) in complex with hSyt I peptide (purple) and ganglioside (yellow). The LC and HN were added from superposition with the full-length BoNT/B (PDB 1EPW). Location of the WW mutations is circled. (B) Overlay of the crystal structures of WT HC/B (gray, PDB 4KBB) and HC/BWW (blue), with the ganglioside oligosaccharide (pink and yellow, from the respective structures) and hSyt I (red)/rSyt II (yellow). The two mutated W residues are shown as sticks. (C) The region containing the GBS, Syt-binding site, and LBL is enlarged, with electron density (Fo-Fc map at 1σ) of GD1a, Syt I, and the two mutated W residues shown as a blue mesh. (D) HC/BWW is modeled onto membranes through anchoring with both GD1a and Syt I, showing that the two W residues in its LBL interact with membranes. BoNT, botulinum neurotoxin; GBS, ganglioside-binding site; GD1a, disialoganglioside; HC, C-terminal receptor-binding domain; HN, N-terminal translocation domain; hSyt I, human Syt I; LBL, lipid-binding loop; LC, light chain; PDB, Protein Data Bank; rSyt II, rat Syt II; Syt, synaptotagmin; WT, wild-type.