The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans. There are seven serotypes of BoNTs (A-G) based on a lack of cross anti-sera neutralization. BoNTs utilize gangliosides as components of the host receptors for binding and entry into neurons. Members of BoNT/C and BoNT/D serotypes include mosaic toxins that are organized in D/C and C/D toxins. One D/C mosaic toxin, BoNT/D-South Africa (BoNT/D-SA), was not fully neutralized by immunization with BoNT serotype /C or /D, which stimulated this study. Here the crystal structures of the receptor binding domains of BoNT/C, BoNT/D, and BoNT/D-SA are presented. Biochemical and cell binding studies show that BoNT/C and BoNT/D-SA possess unique mechanisms for ganglioside binding. These studies provide new information on how the BoNTs can enter host cells as well as a basis for understanding the immunological diversity of these neurotoxins.

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The clostridial neurotoxins (CNTs) are among the most potent protein toxins for humans and are responsible for botulism, a flaccid paralysis elicited by the botulinum toxins (BoNT), and spastic paralysis elicited by tetanus toxin (TeNT). Seven serotypes of Botulinum neurotoxins (A–G) and tetanus toxin showed different toxicities and cleave their substrate with different efficiencies. However the molecular basis of their different catalytic activities to their substrates is not clear. BoNT/B light chain (LC/B) and TeNT light chain (LC/T) cleave Vesicle-Associated Membrane Protein 2 (VAMP2) at the same scissile bond, but possess different catalytic activities and substrate requirements, which make them the best candidates to study the mechanisms of their different catalytic activities. The recognition of five major P sites of VAMP2 (P7, P6, P1, P1′ and P2′) and fine alignment of P2, P3 and P2, P4 sites by LC/B and LC/T respectively, contributed to their substrate recognition and catalysis. Significantly, we found that the S1 pocket mutation LC/T(K168E) increased the rate of native VAMP2 cleavage to approach the rate of LC/B, which explains the molecular basis for the lower kcat that LC/T possesses for VAMP2 cleavage relative to LC/B. This analysis explains the molecular basis underlining the VAMP2 recognition and cleavage by LC/B and LC/T and provides insight that may extend the pharmacologic utility of these neurological reagents.

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Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of Synaptotagmin (Syt)-I and SytII, homologous synaptic vesicle proteins. We observe conditions in which BoNT/B binds both Syt isoforms, but BoNT/G only binds SytI. Both serotypes bind ganglioside G T1b . The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform to understand the physiological relevance of different Syt receptor isoforms in vivo.Botulinum neurotoxin (BoNT) is the causative agent of botulism, a potentially lethal neuroparalytic condition in humans (1). The extreme potency of BoNT (LD 50 value ∼ 0.1 ng kg -1 ) stems from the toxin's high affinity for neuronal receptors at the neuromuscular junction and enzymatic inhibition of neurotransmitter release (2). BoNT's are produced as single chain proteins in seven antigenically distinct forms (serotypes A-G). Most BoNT serotypes undergo post-translational cleavage to form a dichain molecule composed of a light-and heavy-chain linked by a disulfide bond. The light chain (LC) is a zinc metalloprotease that cleaves SNARE proteins to inhibit neurotransmitter vesicle fusion to the plasma membrane (3). The N-terminal half of the heavy chain (HCT) is involved in translocation of the LC across the endosomal membrane, and the C-terminal half of the heavy chain (HCR) is involved in binding receptors (4).BoNT targets the neuromuscular junction through specific interactions with both ganglioside and protein receptors (5). BoNTs bind G D1a and gangliosides in the G 1b series and show the highest affinity for the trisialoganglioside, G T1b (6). The protein receptor can vary with BoNT serotype. Synaptotagmin (Syt)-I and SytII mediate the internalization of BoNT/B and/G, but not BoNT/A or/E, into neuronal cells (7-9). SytI and SytII are homologous calcium sensors that couple neuronal calcium influx to the fast phase of neurotransmitter release (10). BoNT/B and/G bind to the luminal domains of SytI and SytII following the fusion of synaptic vesicles with the plasma membrane. The ability of a peptide, corresponding to 20 amino acids of the SytII luminal domain, in conjunction with gangliosides, to neutralize BoNT/B toxicity in mice is consistent with the SytII luminal domain being the neuronal receptor for BoNT/B (7).* To whom correspondence should be addressed. jtb01@mcw.edu; phone (414) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptBoNT/G is a recently discovered serotype whose HCR shares a high degree of primary amino acid conservation with BoNT/B (50% identical, 71% similar). Despite the homology between BoNT/B and BoNT/G and between SytI and SytII, several differences exist in their interactions with neurons. BoNT/B binds SytII independent of ganglioside but requires gangliosid...