High‐resolution crystal structures of the botulinum neurotoxin binding domains from subtypes A5 and A6

Abstract

Botulinum neurotoxins (BoNTs) cause the deadly condition called botulism, but they can be used as therapeutics for a wide range of indications. BoNTs are classified into many different serotypes and subtypes, each with potentially different intoxication properties. Here, we report crystal structures of the receptor‐binding domains from two subtypes (BoNT/A5 and BoNT/A6) and compare their binding sites with previous BoNT/A structures.

Crystal Structures of Botulinum Neurotoxin Subtypes A4 and A5 Cell Binding Domains in Complex with Receptor Ganglioside

“…), H C /A1:GD1a (PDB: 5TPC [22]), H C /A3 (PDB: 6F0O [22]), H C /A3:GD1a (PDB: 6THY [22]), H C /A4 (PDB: 6F0P [24]), H C /A4:GD1a (this study), H C /A5 (PDB: 6TWP [24]), and H C /A5:GM1b (this study). ) and bound to GD1a (orange; PDB: 5TPC [22]); (B) HC/A3 unbound (green; PDB: 6F0O [24]) and bound to GD1a (dark grey; PDB: 6THY [23]); (C) HC/A4 unbound (burlywood; PDB: 6F0P [24]) and bound to GD1a (cyan; this study); (D) HC/A5 unbound (grey; PDB: 6TWP [25]) and bound to GM1b (cyan; this study). The arrow points to the loop that follows the GBS-β hairpin; dotted lines indicate unmodeled regions of the loop. )…”

“…Structural comparison of the loop that follows the β-hairpin of the ganglioside binding site with and without oligosaccharide. (A) H C /A1 unbound (yellow; PDB: 2VUA[29]) and bound to GD1a (orange; PDB: 5TPC[22]); (B) H C /A3 unbound (green; PDB: 6F0O[24]) and bound to GD1a (dark grey; PDB: 6THY[23]); (C) H C /A4 unbound (burlywood; PDB: 6F0P[24]) and bound to GD1a (cyan; this study); (D) H C /A5 unbound (grey; PDB: 6TWP[25]) and bound to GM1b (cyan; this study). The arrow points to the loop that follows the GBS-β hairpin; dotted lines indicate unmodeled regions of the loop.…”

“…We have previously reported the crystal structures of H C /A4 [24] and H C /A5 [25], and now present the crystal structures of H C /A4 in complex with GD1a, and H C /A5 in complex with GM1b, and highlight the interactions and structural changes that occur upon ganglioside binding. The structural information revealed in this report may aid in the development of future BoNT therapeutics.…”

“…The sequences of BoNT/A4 residues 870-1296 (HC/A4) and BoNT/A5 residues 871-1296 (HC/A5) were cloned into the pJ401 vector with an N-terminal hexa-histidine tag, as described previously [24,25]. Both constructs were transformed into E. coli strain BL21 and grown at 37 • C to an OD600 of 0.5 before induction with 1 mM IPTG for 16 h at 16 • C. Cells were harvested by centrifugation.…”

“…A total of 7200 images were collected for H C /A4:GD1a, and 3600 images for H C /A5:GM1b. Data processing was carried out in DIALS [32] and both structures were solved by molecular replacement in PHASER [33] using a previously reported structure of H C /A4 [24] and H C /A5 [25] as search models. Initial rounds of refinement were performed using REFMAC [26] as part of the CCP4 package [34] with the final round of refinement and validation performed in Phenix [35].…”