Inter-Domain interactions Slow BoNT/A's onset of action

Despite sharing ∼ 43 % sequence identity and structurally similar individual domains, botulinum neurotoxin (BoNT) serotypes A and E have differences in their properties and domain positioning. BoNT/E has a faster onset of action than BoNT/A. This difference is proposed to be due to conformational differences between BoNT/E and the other BoNT serotypes. Where most serotypes have the light chain (LC) and binding domain (BD) on opposite sides of the translocation domain (TD), BoNT/E forms a more compact shape with direct interactions between residues of the LC and BD. To elucidate the structural basis for the different properties of BoNT/A and BoNT/E, biophysical studies including molecular dynamic (MD) simulations, circular dichroism (CD) and small-angle X-ray scattering (SAXS) were applied to BoNT/A, for comparison against previous work on BoNT/E. MD simulationsat six pH values across the toxin's activation barrier (pH ∼ 5.5), followed by one extra repeat for the pH values below 5.5, revealed a rare event at pH 5 and 5.5 where interactions between a previously identified switch region of BoNT/Aand the BD were lost.This hintedat an increased freedom of movement, thus allowing the region to change from α-helical to a β-hairpin. In good agreement with previous work, CD showed a gradual and small loss of helicity as the pH decreased below pH 5.5, stabilising at pH 4.5. Combined with the relative scarcity of structural changes observed by MD in the switch region required for activity, these results may explain the slower onset of action for BoNT/A compared to BoNT/E.