Identification of a unique ganglioside binding loop within botulinum neurotoxins C and D-SA

Abstract

The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans. There are seven serotypes of BoNTs (A-G) based on a lack of cross antiserum neutralization. BoNTs utilize gangliosides as components of the host receptors for binding and entry into neurons. Members of BoNT/C and BoNT/D serotypes include mosaic toxins that are organized in D/C and C/D toxins. One D/C mosaic toxin, BoNT/D-South Africa (BoNT/D-SA), was not fully neutralized by immunization with BoNT serotype C or D, which stimulated this study. Here the crystal structures of the receptor binding domains of BoNT/C, BoNT/D, and BoNT/D-SA are presented. Biochemical and cell binding studies show that BoNT/C and BoNT/D-SA possess unique mechanisms for ganglioside binding. These studies provide new information about how the BoNTs can enter host cells as well as a basis for understanding the immunological diversity of these neurotoxins.

Figure 1. Crystal structures of HCR/C, HCR/D, and HCR/D-SA

(A) Shown are overlays of the crystal structure of the HCR/D-SA (blue) with HCR/C (left panel, red) RMSD: 0.46 and HCR/D-SA (blue) with HCR/D (right panel, green) RMSD: 2.47. (B) Structure-based alignments of the C-terminal subdomains of HCR/C, HCR/D, and HCR/D-SA with HCR/A and HCR/B are shown. β strands, α helix, and loops are numbered from residue 1095 of HCR/C. An internal W and F (W1278 and F1280 of HCR/D-SA), highlighted blue, were conserved among the HCRs and provided a point of reference for the alignment. Residues within the ganglioside binding domain of HCR/A are green (E, H, S, W, and Y). Tryptophan within the ganglioside binding loop of HCR/C (W1258) and HCR/D-SA (W1252) are in pink. A W, located at a different position in the GBL of HCR/D (W1238), is pink. Yellow colored residues indicate conserved amino acids within the HCRs that have not been associated ganglioside or receptor binding functions.

Figure 1. Crystal structures of HCR/C, HCR/D, and HCR/D-SA

(A) Shown are overlays of the crystal structure of the HCR/D-SA (blue) with HCR/C (left panel, red) RMSD: 0.46 and HCR/D-SA (blue) with HCR/D (right panel, green) RMSD: 2.47. (B) Structure-based alignments of the C-terminal subdomains of HCR/C, HCR/D, and HCR/D-SA with HCR/A and HCR/B are shown. β strands, α helix, and loops are numbered from residue 1095 of HCR/C. An internal W and F (W1278 and F1280 of HCR/D-SA), highlighted blue, were conserved among the HCRs and provided a point of reference for the alignment. Residues within the ganglioside binding domain of HCR/A are green (E, H, S, W, and Y). Tryptophan within the ganglioside binding loop of HCR/C (W1258) and HCR/D-SA (W1252) are in pink. A W, located at a different position in the GBL of HCR/D (W1238), is pink. Yellow colored residues indicate conserved amino acids within the HCRs that have not been associated ganglioside or receptor binding functions.

Figure 2. Overlay of the Ganglioside Binding Pocket of HCR/A with the representative residues of HCR/D-SA

HCR/D-SA (blue) is shown with the following highlighted residues: conserved internal F1280 and W1282 (grey), and corresponding residues that represent the Ganglioside Binding Pocket of HCR/A (green). Ganglioside Binding Pocket of HCR/A (lower) and the corresponding region of HCR/D-SA (upper) were expanded and shown. Residues that contribute to ganglioside binding of HCR/A (E1203, H1253, S1264, W1266 and Y1267) and corresponding residues within HCR/D-SA that align in space are shown in green with nitrogen and oxygen atoms depicted in blue and red respectively. This alignment indicates the major determinant of ganglioside binding, a conserved tryptophan residue present in the ganglioside pocket of BoNT/A, /B, /E, /F, /G and TeNT is absent from the structurally analogous region of HCR/D-SA.

Figure 3. Overlay of the Ganglioside Binding Loops of HCR/C, HCR/D, and HCR/D-SA with the representative loops of HCR/A and HCR/B

HCR/D-SA (blue, upper) is shown with the conserved, buried F1280 and W1282 (black), and the Ganglioside Binding Loop (GBL) W1252 indole ring is also shown at the tip of the loop. The GBL was enlarged, rotated, and shown (lower) aligned with the structurally analogous β-hairpin loops of BoNT/A (purple), /B (orange), /C (red), /D (green). HCR/C and /D-SA loops display a similar overall structural arrangement with the indole ring of the W residue implicated in ganglioside binding (HCR/C (W1258), HCR/D-SA (W1252)) extending away from the HCR molecule. HCR/D, like HCR/C and /D-SA has a tryptophan residue (W1238) in the β-hairpin loop. However, unlike HCR/C and /D-SA, the HCR/D W1238 indole ring does not extend away from the HCR but rather is oriented toward an adjacent β-hairpin loop. BoNT/B has an extended β-hairpin loop like HCR/C, /D, and /D-SA but lacks a tryptophan residue. BoNT/A, in contrast, does not have an extended β-hairpin loop.

Figure 4. HCR/C and HCR/D-SA binding to complex gangliosides

Wells (96 well plates) were coated with the indicated ganglioside (0.5 μg per well in 100 μl of MeOH). Plates were dried and 10 nM of HCR/C, HCR/D-SA, or HCR/T was incubated in 100 μl of PBS for 1 hr at 4°C. Wells were washed and bound HCR was detected with anti-FLAG-HRP antibody followed by TMB-Ultra. A450 was determined as the average of three independent experiments.

Figure 5. Role of Tryptophan within the ganglioside binding loop in HCR/C and HCR/D-SA binding to complex gangliosides

Wells (96 well plates) were coated with GD1b (upper panel) or GM1a (lower panel) (0.5 μg (HCR/C) or 2.0 μg (HCR-D-SA) per well in 100 μl of MeOH). Plates were dried and 10 nM of HCR/C or HCR/C (W1258A) (upper panel) or HCR/D-SA or HCR/D-SA (W1252A) (lower panel) was incubated in 100 μl of buffer for 1 hr at 4°C. Bound HCR was detected with an M2 anti-FLAG antibody followed by TMB-Ultra. A450 was determined and plotted as the average of at least duplicate determinations. Background absorbance of HCR bound to well in the absence of ganglioside was subtracted to account for non-specific binding. No binding above background was detected for HCR/D-SA (W1252A) so these values are presented as zero.

Figure 6. Crystal structure of HCR/D-SA(W1252A)

Overlay of the crystal structures of the C-terminal subdomains (residues 1091-1285) of HCR/D-SA(W1252A) (orange) and HCR/D-SA (blue) are shown. The indole ring (yellow) of W1252 of HCR/D-SA and the methyl group (pink) of A1252 of HCR/D-SA(W1252A) are also shown.

Figure 7. Binding of HCR/C, HCR/D, and HCR/D-SA to primary cortical neurons

Primary cortical neurons were plated and cultured in 24-well glass bottom culture dishes coated with poly-D-lysine and laminin. Following 10 days of culture, cells were washed and incubated with a dilution series of the indicated HCR in media for 1 hr at 4°C. Bound HCR was identified by immunofluorescence microscopy using mouse anti-Flag mAb followed by goat anti-mouse IgG-488. A) Representative images of the indicated HCR (40 nM) bound to neurons (left panels) and nuclei stained with Hoechst (right panels) are shown (60X magnification). Images were captured at identical exposure times, and analyzed using Image J. Scale Bar = 35μm. B) Quantification of HCR binding to neurons. The values presented are arbitrary fluorescence units and represent the average FLAG fluorescence intensity from five random fields following correction for non-specific fluorescence. The average of two independent experiments is shown.