The 25 kDa H(CN) Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity

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 H(N), and the C-terminal receptor binding domain H(CC) are largely resolved, but that of the H(CN) domain sandwiched between H(N) and H(CC) has remained unclear. Here, mutants of BoNT/A, BoNT/B, and TeNT were generated by deleting their H(CN) domains or swapping H(CN) domains between each other. Both deletion and replacement of TeNT H(CN) domain by H(CN)A and H(CN)B 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 H(CN) domains between BoNT/A and B hardly impaired their biological activity, but substitution with H(CN)T did. Binding assays revealed that in the absence of H(CN), not all receptor binding sites are equally well accessible. In conclusion, the presence of H(CN) is vital for CNTs to exert their neurotoxicity. Although structurally similar, the H(CN) domain of TeNT cannot equally substitute those of BoNT and vice versa, leaving the possibility that H(CN)T plays a different role in the intoxication mechanism of TeNT.