Identification of the receptor-binding sites in the carboxyl-terminal half of the heavy chain of botulinum neurotoxin types C and D

Botulinum neurotoxin (BoNT) binds to presynaptic neuronal cells and blocks neurotransmitter release. The carboxyl-terminal half of the heavy chain (H_C) of the neurotoxin recognizes its specific receptor on the plasma membrane. We have previously demonstrated that BoNT/C binds to gangliosides GD1b and GT1b under physiological conditions, while BoNT/D interacts with phosphatidylethanolamine (PE). Here we report that the recognition sites for gangliosides and PE are present in the carboxyl-terminal domain of H_C. Chimeric mutants and site-directed mutants of BoNT/C-H_C and BoNT/D-H_C were generated and their binding activities evaluated. The chimeric H_C that consisted of the amino-terminal half of BoNT/D-H_C and the carboxyl-terminal half of BoNT/C-H_C possessed activity similar to the authentic BoNT/C-H_C, suggesting that the carboxyl-terminal region of H_C is involved in the receptor recognition of BoNT/C. Moreover, analysis using site-directed mutants indicated that the peptide motif W¹²⁵⁷Y⋯G¹²⁷⁰⋯H¹²⁸² plays an important role in the interaction between BoNT/C and gangliosides. In contrast, we revealed that two lysine residues of BoNT/D-H_C are involved in the formation of the critical binding site for receptor binding.