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The extraordinarily potent clostridial neurotoxins (CNTs) comprise tetanus neurotoxin (TeNT) and the seven established botulinum neurotoxin serotypes (BoNT/A-G). They are composed of four structurally independent domains: the roles of the catalytically active light chain, the translocation domain HN, and the C-terminal receptor binding domain HCC are largely resolved, but that of the HCN domain sandwiched between HN and HCC has remained unclear. Here, mutants of BoNT/A, BoNT/B, and TeNT were generated by deleting their HCN domains or swapping HCN domains between each other. Both deletion and replacement of TeNT HCN domain by HCNA and HCNB reduced the biological activity similarly, by ~95%, whereas BoNT/A and B deletion mutants displayed >500-fold reduced activity in the mouse phrenic nerve hemidiaphragm assay. Swapping HCN domains between BoNT/A and B hardly impaired their biological activity, but substitution with HCNT did. Binding assays revealed that in the absence of HCN, not all receptor binding sites are equally well accessible. In conclusion, the presence of HCN is vital for CNTs to exert their neurotoxicity. Although structurally similar, the HCN domain of TeNT cannot equally substitute those of BoNT and vice versa, leaving the possibility that HCNT plays a different role in the intoxication mechanism of TeNT. [ABSTRACT FROM AUTHOR]