Activity of botulinum neurotoxin X and its structure when shielded by a non-toxic non-hemagglutinin protein

Abstract

Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex and the crystal structure of the isolated NTNH protein. Unexpectedly, the BoNT/X complex is stable and protease-resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency both in vitro and in vivo. Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents very weak ganglioside binding and exposed hydrophobic surfaces.

Fig. 1. Activity of BoNT/X.

Rat cortical neurons were exposed to varying concentrations of recombinant full-length BoNT/X (filled circles) or LH_NX (open circles). After 24 h of incubation cells were lysed, and lysates analyzed for VAMP2 (a) and VAMP4 (b) by Western blots (error bars represent standard deviation from n = 3 replicates). c Mouse phrenic nerve hemidiaphragm (mPNHD) preparations were exposed to 10 pM BoNT/X, BoNT/A, and BoNT/B, 100 pM LH_N/X, and muscle contraction responses to indirect stimulation recorded until contraction was no longer detectable or to 180 min (n = 3). d BoNT/A (6 pg) induced flaccid paralysis when injected into the right gastrocnemius muscle of mice in DAS assays, whereas injection of BoNT/X (1 µg) did not induce muscle paralysis. e LH_N/X (40 µg) was injected to the right gastrocnemius muscle of mice (n = 3). The limb developed partial paralysis after 24 h (DAS Score = 2 out of 4) and the toes showed a reduced ability to spread following a startle stimulus. LH_N/X that had not been activated with trypsin served as a control. A representative image of paralysis obtained from injection of 40 µg is shown and experiment data are shown in Supplementary Table 4.

Fig. 2. Cryo-EM structure of BoNT/X in complex with NTNH/X.

a Surface view of the BoNT/X (green)—NTNH (blue) complex. b Cartoon view of the different domains in each protein. BoNT/X domains are colored in green shades, NTNH/X domains are colored in blue shades. c Local resolution estimates (Å) for the cryo-EM map of the M-PTC/X. d cryo-EM map around the LC active site. e cryo-EM map around the SxWY ganglioside-binding motif. f cryo-EM map around the H_C patch rich in aromatic residues.

Fig. 3. Structure of the BoNT/X light chain.

a H_N and LC domains from the BoNT/X-NTNH/X cryo-EM structure (green). The belt region of H_N surrounding the LC is highlighted in orange. b crystal structure of LC/F (PDB ID 3FIE, colored in red) bound to a VAMP substrate-like inhibitor (purple) superimposed to H_N-LC/X (green). c, d crystal structure of LC/X (PDB ID 6F47, colored in magenta) superimposed to the cryo-EM structure of H_N-LC/X (green). Noteworthy differences in secondary structure are highlighted in (d).

Fig. 4. BoNT/X-NTNH/X complex stability.

a BoNT/X-NTNH/X interface; BoNT/X is shown in green and NTNH/X in blue. Top: interfacing residues are highlighted in different shades of green and blue. Bottom: residues at the interface of the complex are colored according to electrostatic potential (negative electrostatic potential is colored in shades of red, and positive in shades of blue). b Electrostatic interactions across the BoNT/X-NTNH/X interface. c Size-exclusion chromatograms from the M-PTC/X pH stability experiments. The A_280nm response for the chromatograms at pH 5.5 and 6.5 was divided by the factor of two to normalize it to the other chromatograms, where the signal was lower. d Flexible linker between H_N and H_C followed by a small helical region leading to the H_C (residues 876–895). e, f Limited proteolysis assay for examining the stability of the BoNT/X-NTNH/X complex at different pH conditions. BoNT/X (marked as X), NTNH/X (marked as N), and BoNT/X-NTNH/X complex (marked as XN) were incubated with trypsin at the indicated pH conditions and then analyzed by SDS-PAGE and visualized by Coomassie blue staining. BoNT/X alone and NTNH/X alone were degraded at all pH conditions. BoNT/X-NTNH/X is largely resistant to trypsin at pH 5.0, 6.0, and 7.5, but not at pH 8.0. Trypsin treatment is able to cleave the linker between LC and HC of BoNT/X within the BoNT/X-NTNH/X complex, thus BoNT/X showed as two separate bands (LC/X and HC/X) after the disulfide bond connecting the LC and HC is reduced. One contaminant is present in purified BoNT/X and another is present in purified BoNT/X-NTNH/X complex.

Fig. 5. Investigation of potential ganglioside binding by BoNT/X.

a Alignment of H_C/X (green) and GD1a-bound H_C/A (in grey, PDB ID 5TPC, GD1a shown in grey sticks), showing the conserved ganglioside-binding SxWY motif residues and (b) the same alignment showing the exposed region rich in aromatic residues. Location of the ganglioside-binding motif (yellow) and the hydrophobic patch (orange) is shown in the context of H_c/X in (c). Binding of BoNT/X (green), and H_C/A (grey) to GT1b (d), GD1a (e), GM1 (f), and GD1b (g), error bars represent standard deviation from n = 3 replicates. Glycobloc schematic representation is shown for each ganglioside.