Unique ganglioside recognition strategies for clostridial neurotoxins

Abstract

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin are the causative agents of the paralytic diseases botulism and tetanus, respectively. The potency of the clostridial neurotoxins (CNTs) relies primarily on their highly specific binding to nerve terminals and cleavage of SNARE proteins. Although individual CNTs utilize distinct proteins for entry, they share common ganglioside co-receptors. Here, we report the crystal structure of the BoNT/F receptor-binding domain in complex with the sugar moiety of ganglioside GD1a. GD1a binds in a shallow groove formed by the conserved peptide motif E … H … SXWY … G, with additional stabilizing interactions provided by two arginine residues. Comparative analysis of BoNT/F with other CNTs revealed several differences in the interactions of each toxin with ganglioside. Notably, exchange of BoNT/F His-1241 with the corresponding lysine residue of BoNT/E resulted in increased affinity for GD1a and conferred the ability to bind ganglioside GM1a. Conversely, BoNT/E was not able to bind GM1a, demonstrating a discrete mechanism of ganglioside recognition. These findings provide a structural basis for ganglioside binding among the CNTs and show that individual toxins utilize unique ganglioside recognition strategies.

FIGURE 1.

Wild-type HCR binding kinetics. Various concentrations of recombinant HCRs were examined for their ability to bind GD1a: HCR/A (●), HCR/E (■), and HCR/F (▴). B₅₀ values represent half-maximal binding to the ganglioside. All values represent the arithmetic mean ± S.D. of at least four independent experiments performed in triplicate.

FIGURE 2.

Structure of HCR/F in complex with the GD1a-OS. A, schematic representation of HCR/F shown in green. The GD1a-OS is shown as atomic color sticks. B, σ_A weighted F_o − F_c omit map of the GD1a-OS contoured at 3.0σ. Electron density for glucose 1 (shown in gray) is not well defined. C, ribbon representation of the complex illustrating the positioning of the GD1a-OS in the ganglioside-binding cleft and the intermolecular hydrogen bond interactions. The two bridging water molecules are shown as red spheres. Residues not previously identified as contributing to ganglioside binding are highlighted asterisks.

FIGURE 3.

Two arginine residues facilitate ganglioside binding at the Sia5 position. Various concentrations of recombinant HCR/F proteins were examined for their ability to bind GD1a: wild type (▴), R1111A (▾), R1256A (♦), and R1111A,R1256A (♢). B₅₀ values represent half-maximal binding to ganglioside. All values represent the arithmetic mean ± S.D. of two independent experiments performed in triplicate.

FIGURE 4.

Superposition of the HCR/E and HCR/F ganglioside-binding pockets. Shown is a ribbon representation of HCR/F overlaid with the isolated HCR of BoNT/E (Protein Data Bank code 3FFZ). Residues constituting the conserved GBM of HCR/F and HCR/E are shown (stick representation). The key Arg-1111, His-1241, and Arg-1256 residues of HCR/F and the corresponding residues of HCR/E are highlighted (stick representation, atomic color). The altered loop region for HCR/F (Ile-1255–Asn-1262) is shown in green, and that for HCR/E (Met-1229–Asn-1236) is in yellow.

FIGURE 5.

Comparative analysis of HCR binding to gangliosides GD1a and GM1a. Various concentrations of the recombinant HCRs were examined for their ability to bind GD1a. A, HCR/E (■), HCR/E^K1215A (♢), and HCR/E^K1215H (□). B, HCR/F (▴) and HCR/F^H1241K (△). C, HCR/F^{R1111A,H1241K,R1256A} (○) and HCR/F^{R1111A,R1256A} (●; compared using data from Fig. 3). D, various concentrations of recombinant HCR/E (■), HCR/F (▴), and HCR/F^H1241K (△) were examined for their ability to bind GM1a. B₅₀ values represent half-maximal binding to ganglioside. Values represent the arithmetic mean ± S.D. of three independent experiments performed in triplicate.

FIGURE 6.

Mutational analysis of the lactose-binding site in the tetanus receptor-binding domain (HCR/T). The lactose-binding binding site in HCR/T is structurally similar to the ganglioside-binding site in the botulinum HCRs. Various concentrations of the recombinant wild-type and variant HCRs for HCR/T^R1226L (○), HCR/F (▴), and HCR/T^{N1219A,R1226L} (●) were examined for their ability to bind GD1a. B₅₀ values represent half-maximal binding to ganglioside. All values represent the arithmetic mean ± S.D. of two independent experiments performed in triplicate.

FIGURE 7.

Schematic of HCR-mediated ganglioside binding. In each of the panels, monosaccharides are represented by hexagons. GalNAc3 and Gal4 are colored in light gray, whereas Sia5 is colored in dark gray. HCR amino acid residues involved in ganglioside binding are represented using their single-letter codes. Hydrogen bonds are indicated by dotted lines between the amino acid in question and the indicated sugar.