Botulinum Neurotoxin A4 Has a 1000-Fold Reduced Potency Due to Three Single Amino Acid Alterations in the Protein Receptor Binding Domain

Botulinum neurotoxin subtype A4 (BoNT/A4) is ~1000-fold less potent than BoNT/A1. This study addresses the basis for low BoNT/A4 potency. Utilizing BoNT/A1-A4 and BoNT/A4-A1 Light Chain-Heavy Chain (LC-HC) chimeras, HC-A4 was responsible for low BoNT/A4 potency. Earlier studies showed BoNT/A1-receptor binding domain (Hcc) bound a β-strand peptide (556–564) and glycan-N(559) within Luminal Domain 4 (LD4) of SV2C, the BoNT/A protein receptor. Relative to BoNT/A1, the Hcc of BoNT/A4 possesses two amino acid variants (D(1141) and N(1142)) within the β-peptide binding interface and one amino acid variant (R(1292)) located near the SV2C glycan-N(559). Introduction of BoNT/A4 β-strand peptide variant (D(1141) and N(1142)) into BoNT/A1 reduced toxin potency 30-fold, and additional introduction of the BoNT/A4 glycan-N(559) variant (D(1141), N(1142), and R(1292)) further reduced toxin potency to approach BoNT/A4. While introduction of BoNT/A1 glycan-N(559) variant (G(1292)) into BoNT/A4 did not alter toxin potency, additional introduction of BoNT/A1 β-strand peptide variants (G(1141), S(1142), and G(1292)) resulted in potency approaching BoNT/A1 potency. Thus, outcomes from these functional and modeling studies indicate that in rodent models, disruption of Hcc -SV2C β-peptide and -glycan-N(559) interactions mediate low BoNT/A4 potency, while in human motor neurons, disruption of Hcc-SV2C β-peptide alone mediates low BoNT/A4 potency, which link to a species-specific variation at SV2C(563).