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

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 (H C ), the translocation domain (H N ), and the catalytic domain (LC). The H C, which consists of an N-terminal (H CN ) and a C-terminal (H CC ) 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 (H C /A6) in complex with GD1a and describe the interactions involved in ganglioside binding. We also present a new crystal form of wild type H C /A6 (crystal form II) where a large 'hinge motion' between the H CN and H CC subdomains is observed. These structures, along with a comparison to the previously determined wild type crystal structure of H C /A6 (crystal form I), reveals the degree of conformational flexibility exhibited by H C /A6.