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_CNA and H_CNB 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_CNT 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_CNT plays a different role in the intoxication mechanism of TeNT.