Fig 3. Introducing aromatic residues at the tip of H_CB-LBL enables its binding to lipid membranes.

(A) Structure of H_CB-LBL, with three residues at the tip marked. (B–C) Binding of H_C/B mutants containing either a single point mutation, I1248W, or double point mutations, I1248W/V1249W, to ND1 were analyzed by BLI assays. H_C/B^W showed low-level binding to ND1, whereas H_C/B^WW showed robust binding. (D) The maximal binding signals of H_C/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 H_C/B, H_C/B^W, and H_C/B^WW 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. H_C/B^W and H_C/B^WW 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; H_C, 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.