Structural basis of botulinum neurotoxin serotype A1 binding to human SV2A or SV2C receptors.

Botulinum neurotoxin A1 (BoNT/A1) is the most potent natural poison in human. BoNT/A1 recognize the luminal domain of SV2A (LD-SV2A) and its glycosylation at position N573 (N573g) or the luminal domain of SV2C (LD-SV2C) and its glycosylation at position N559 (N559g) to bind neural membrane. Our computational data suggest that the N-glycan at position 480 (N480g) in the luminal domain of SV2C (LD-SV2C) indirectly enhanced the contacts of the neurotoxin surface with the second N-glycan at position 559 (N559g) by acting as a shield to prevent N559g to interact with residues of LD-SV2C. The absence of an N-glycan homologous to N480g in LD-SV2A leads to a decrease of the binding of N573g to the surface of BoNT/A1. Concerning the intermolecular interactions between BoNT/A and the protein part of LD-SV2A or LD-SV2C, we showed that the high affinity of the neurotoxin for binding LD-SV2C are mediated by a better compaction of its F557-F562 part provided by a π-π network mediated by residues F547, F552, F557 and F562 coupled with the presence of two aromatic residues at position 563 and 564 that optimize the binding of BoNT/A1 via cation-pi and CH-pi interaction. Finally, in addition to the well-known ganglioside binding site which accommodates a ganglioside on the surface of BoNT/A1, we identified a structure we coined the ganglioside binding loop defined by the sequence 1253-HQFNNIAK-1260 that is conserved across all subtypes of BoNT/A and is predicted to has a high affinity to interact with gangliosides. These data solved the puzzle generated by mutational studies that could be only partially understood with crystallographic data that lack both a biologically relevant membrane environment and a full glycosylation of SV2.