Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity

Abstract

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.

Figure 1. The molecular architecture of L-PTC/A.

(A) 3D-EM reconstruction of L-PTC/A. Structural models of the M-PTC and the HA complex were fit into the EM envelope. The red arrow indicates the viewing direction of (C). (B) Surface representation of L-PTC/A in the same orientation as (A). (C) A different view of L-PTC/A. (D) An open-book view of the interface that is highlighted in the box in (B). See Fig. S2 in Text S1 for stereo versions of (A) and (B).

Figure 2. X-ray structures of the HA complex.

(A) Structure of the HA70^D3–HA17 complex at 2.4 Å resolution. (B) Structure of the HA17–HA33 complex at 2.1 Å resolution. (C) Structure of HA70 at 2.9 Å resolution. The red arrow indicates the viewing direction of (E). (D) Structure of the mini-HA complex (HA70^D3–HA17–HA33) at 3.7 Å resolution. (E) 3D-EM reconstruction of the complete HA complex. The structure model of the HA complex was fit into the EM envelope. Open-book views of the interfaces highlighted in the green, orange, and yellow boxes are shown in Fig. 3.

Figure 3. The 12-subunit HA complex is stabilized by extensive protein–protein interactions among three HAs.

Interacting residues are labeled in open-book views of the interfaces. (A) Interface between HA70 and HA17. (B) Interfaces between HA17 and the two HA33s are indicated by purple and blue. The HA33 residues involved in both interfaces are in green. (C) Interface between two HA33s attached to the same HA17. See Fig. S5 and S6 in Text S1 for stereo versions of the detailed interactions.

Figure 4. The fully assembled HA complex markedly reduced the TER of human intestinal Caco-2 cell monolayers.

Caco-2 cells were grown on transwell filter membranes into confluent polarized monolayers. (A, B) TER was measured following application of the L-PTC, the HA complex, the M-PTC, BoNT/A, or NTNHA-A to the apical (A; 58 nM) or basolateral (B; 17 nM) chambers. (C, D) TER was measured when the HA complex (HA wt), the mini-HA complex (HA70^D3–HA17–HA33), HA70 trimer, HA33, or the HA17–HA33 complex were applied to the apical (C; 58 nM) or basolateral (D; 17 nM) chambers. Values are means ± SD (n = 4–12).

Figure 5. The HA complex interacts with carbohydrate receptors to cross epithelial cell monolayers.

(A, B) TER of Caco-2 monolayers was measured when Alexa-488-labeled HA complex (HA*) pre-incubated with Lac, IPTG, α2,3-SiaLac, or α2,6-SiaLac was applied to the apical (A; 58 nM) or basolateral (B; 17 nM) chambers. Values are means ± SD (n = 4–12). (C) HA* (with or without carbohydrates) or the Alexa-488-labeled HA^33-DAFA complex (HA^33-DAFA *) was applied to the apical (at 58 nM) or basolateral (at 17 nM) chamber. The fluorescence signals in both chambers were quantified after 24 hours and the amount of transported HA*/HA^33-DAFA * was expressed as a percentage of the total HA*/HA^33-DAFA * used. Values are means ± SD (n = 3–22).

Figure 6. The HA complex mediates BoNT absorption through multivalent interactions with glycan receptors.

Close-up views of HA70–α2,3-SiaLac and HA33–Lac interactions are shown in (A) and (B), respectively. Key HA residues involved in glycan coordination are shown as sticks. Hydrogen bonds are indicated by black dashed lines. (C) The HA complex has nine glycan-binding sites. (D, E) TER of the Caco-2 monolayers were measured after application of the wild-type HA complex (HA wt), the HA^70-TPRA complex, or the HA^33-DAFA complex to the apical (D) or basolateral (E) chambers. Values are means ± SD (n = 4–12). (F) Survival comparisons of mice treated orally with L-PTC/A in the presence or absence of IPTG, Neu5Ac, and Gal.