Assembly and function of the botulinum neurotoxin progenitor complex

Abstract

Botulinum neurotoxins (BoNTs) are among the most poisonous substances known to man, but paradoxically, BoNT-containing medicines and cosmetics have been used with great success in the clinic. Accidental BoNT poisoning mainly occurs through oral ingestion of food contaminated with Clostridium botulinum. BoNTs are naturally produced in the form of progenitor toxin complexes (PTCs), which are high molecular weight (up to ~900 kDa) multiprotein complexes composed of BoNT and several non-toxic neurotoxin-associated proteins (NAPs). NAPs protect the inherently fragile BoNTs against the hostile environment of the gastrointestinal (GI) tract and help BoNTs pass through the intestinal epithelial barrier before they are released into the general circulation. These events are essential for ingested BoNTs to gain access to motoneurons, where they inhibit neurotransmitter release and cause muscle paralysis. In this review, we discuss the structural basis for assembly of NAPs and BoNT into the PTC that protects BoNT and facilitate its delivery into the bloodstream.

Fig. 1

The structure of the M-PTC of BoNT/A. BoNT/A domains are blue (LC), orange (H_N), and green (H_C). NTNHA-A domains are yellow (nLC), cyan (nH_N), and red (H_C). The cartoon and surface representations of the M-PTC are shown in the left and right panels, respectively.

Fig. 2

Structures of BoNT/A and NTNHA-A in the M-PTC. The domain colors are as described for Figure 1.

Fig. 3

The H_C of BoNT/A can adopt multiple conformations. Surface representations of the structures of BoNT/A in the M-PTC (a), free forms of BoNT/A (BoNT/B adopts an identical conformation) (b), and free BoNT/E (c). (d) Cartoon model showing the three conformations of the H_C fragment depicted in (a)–(c). The receptor-binding site in H_C is indicated by a red star. (e) Sequence alignment of the H_N-H_C linker. Residues that adopt an α-helix or β-sheet are in orange or green, respectively.

Fig. 4

NTNHA binding does not directly block the receptor-binding sites in BoNT/A. (a) The structure of H_CA in the M-PTC (complex, green) is superimposed with an H_CA–GT1b (apo, orange) complex (PDB code: 2VU9). Key residues that interact with GT1b (purple sticks) are shown in stick representation. Syt-II (ribbon in purple) is modeled based on H_CB–Syt-II complex (PDB code: 2NM1). (b) The structures in panel (a) are shown in the context of the M-PTC with NTNHA-A in a surface representation (gray).

Fig. 5

A model of the assembly of the PTC. The structures are shown in a cartoon representation: HA-17 (salmon, BoNT/D, PDB code 2E4M), HA-33 (green, BoNT/D, PDB code 2E4M), HA-70 (cyan/yellow/gray, BoNT/C, PDB code 2ZS6), BoNT (gold, BoNT/A1, PDB code 3V0A), and NTNHA (blue, BoNT/A1, PDB code 3V0A).