Crystal Structures of the Clostridium botulinum Neurotoxin A6 Cell Binding Domain Alone and in Complex with GD1a Reveal Significant Conformational Flexibility

Abstract

Clostridium botulinum neurotoxin A (BoNT/A) targets the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, by cleaving synaptosomal-associated protein of 25 kDa size (SNAP-25). Cleavage of SNAP-25 results in flaccid paralysis due to repression of synaptic transmission at the neuromuscular junction. This activity has been exploited to treat a range of diseases associated with hypersecretion of neurotransmitters, with formulations of BoNT/A commercially available as therapeutics. Generally, BoNT activity is facilitated by three essential domains within the molecule, the cell binding domain (HC), the translocation domain (HN), and the catalytic domain (LC). The HC, which consists of an N-terminal (HCN) and a C-terminal (HCC) subdomain, is responsible for BoNT’s high target specificity where it forms a dual-receptor complex with synaptic vesicle protein 2 (SV2) and a ganglioside receptor on the surface of motor neurons. In this study, we have determined the crystal structure of botulinum neurotoxin A6 cell binding domain (HC/A6) in complex with GD1a and describe the interactions involved in ganglioside binding. We also present a new crystal form of wild type HC/A6 (crystal form II) where a large ‘hinge motion’ between the HCN and HCC subdomains is observed. These structures, along with a comparison to the previously determined wild type crystal structure of HC/A6 (crystal form I), reveals the degree of conformational flexibility exhibited by HC/A6.

A Comprehensive Structural Analysis of Clostridium botulinum Neurotoxin A Cell-Binding Domain from Different Subtypes

“…Binding occurs within a β-hairpin at the C-terminus of the H CC subdomain ( Figure 4 and Figure 6 ) [ 85 , 86 , 87 , 88 , 89 , 90 ], a region referred to as the ganglioside binding site (GBS), which is formed partly by a conserved ‘H…SxWY…G’ peptide motif [ 67 ] ( Figure 3 and Figure 6 ).…”

“…The H C /A1:GT1b, H C /A1:GD1a, H C /A2:GD1a, H C /A3:GD1a, H C /A4:GD1a, H C /A5:GM1b, and H C /A6:GD1a structures [ 85 , 86 , 87 , 88 , 89 , 90 ] ( Table 1 ) have revealed the precise molecular interactions present across the H C /A:ganglioside interface. Six binding residues across the subtypes analysed here (H C /A1 to H C /A6) are conserved ( Figure 6 A), with some variation depending on the ganglioside.…”

“…In addition, H C /A1 forms 15 hydrogen bonds (including water-mediated hydrogen bonds) with GT1b, but only 10 with GD1a [ 89 , 90 ] ( Figure 6 A). Following on from this, the relative affinity that each H C /A subtype has for GD1a may be inferred from the number of hydrogen bonds present across the interface, which suggests that H C /A1 and H C /A2 possess the highest affinity for GD1a (A1/A2 > A3/A4 > A6) [ 85 , 86 , 87 , 88 , 89 ]. Interestingly, two separate investigations of H C /A1 ganglioside binding showed variable affinity for GD1a—0.6 μM for the entire GD1a molecule [ 95 ] and 1 μM for the sugar moiety only [ 89 ].…”

“…Subsite ‘A’ is occupied by Sia 5 but the orientation of this sugar group within the subsite varies across the H C /A subtypes. In the H C /A2:GD1a, H C /A3:GD1a, H C /A4:GD1a, H C /A5:GM1b, and H C /A6:GD1a structures ( Table 1 ), Sia 5 is oriented perpendicular to Gal 4 , whereas in H C /A1:GD1a it is linear [ 85 , 86 , 87 , 88 , 89 , 90 ] ( Figure 6 B). This difference in Sia 5 positioning can be attributed to several changes across the subtypes.…”

“…The wealth of structural data available on BoNT/A subtype cell-binding domains (H C /A1 to H C /A6) alone and in complex with their receptors have revealed features related to SV2 and ganglioside receptor binding [ 37 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 ]. In particular, the structure of H C /A1 in complex with human glycosylated SV2C [ 37 ] and of H C /A1 to H C /A6 in complex with the ganglioside GD1a (GM1b for H C /A5) [ 85 , 86 , 87 , 88 , 89 , 90 ] ( Table 1 ) have identified six structural features that appear to be common to the cell-binding domain ( Figure 4 ).…”