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

Botulinum neurotoxin A1 (BoNT/A1) is the most potent serotype in humans with the highest clinical duration. BoNT/A1 interacts with synaptic vesicle glycoprotein 2 (SV2) and gangliosides to be taken up by neurons. In this study, we present three molecular dynamics simulations in which BoNT/A1 is in complex with singly or doubly glycosylated SV2C or singly glycosylated SV2A, in a ganglioside rich (lipid raft) context. 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 N-glycosylation at the position N573 (N573g) in the luminal domain of SV2A has a slightly lower affinity for the surface of BoNT/A1 compared to 559g because of possible intermolecular contacts between N573g and residues of the luminal domain of SV2A (LD-SV2A). In addition to the ganglioside binding site (GBS) conserved across serotypes B, E, F and G, the lipid-raft associated GT1b interacted with a structure we coined the ganglioside binding loop (GBL) which is homologous to the lipid binding loop (LBL) in serotypes B, C, D, D/C and G. Finally, we proposed a global model in which BoNT/A1 interacts with its glycosylated protein receptor, one molecule of GT1b interacting in the GBS and five molecules of GT1b interacting with the GBL and residue Y1133. 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. Brief statement We propose a full molecular description of the initial binding of a microbial toxin (Botulinum neurotoxin A1) to the surface of neural cells. Our model includes a protein receptor (SV2) in its native environment, i.e. the periphery of a cluster of gangliosides belonging to a membrane microdomain (lipid raft). A major outcome of our study is the elucidation of the role of the full length glycans (previously resolved by MS spectroscopy) covalently attached to the protein receptor. 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. ### Competing Interest Statement The authors have declared no competing interest.