Botulinum neurotoxin: unique folding of enzyme domain of the most-poisonous poison

Abstract

Botulinum neurotoxin (BoNT), the most toxic substance known to mankind, is the first example of the fully active molten globule state. To understand its folding mechanism, we performed urea denaturation experiments and theoretical modeling using BoNT serotype A (BoNT/A). We found that the extent of BoNT/A denaturation from the native state (N) shows a nonmonotonic dependence on urea concentration indicating a unique multistep denaturation process, N → I₁ I₂ U, with two intermediate states I₁ and I₂. BoNT/A loses almost all its secondary structure in 3.75 M urea (I₁), yet it displays a native-like secondary structure in 5 M urea (I₂). This agrees with the results of theoretical modeling, which helped to determine the molecular basis of unique behavior of BoNT/A in solution. Except for I₂, all the states revert back to full enzymatic activity for SNAP-25 including the unfolded state U stable in 7 M urea. Our results stress the importance of structural flexibility in the toxin’s mechanism of survival and action, an unmatched evolutionary trait from billion-year-old bacteria, which also correlates with the long-lasting enzymatic activity of BoNT inside neuronal cells. BoNT/A provides a rich model to explore protein folding in relation to functional activity.

Keywords:

• botulinum neurotoxin
• protein folding
• MD simulations
• urea denaturation

Acknowledgments

The work was supported in part by US Army Medical Research Activity (Grant USAMRAA-W81XWH-08-P-0705 to B.R.S), and National Institutes of Health (Grant 1U01A1078070-01 to B.R.S). We would like to thank Mr Stephen Riding for preparing protein samples. We would like to thank the anonymous reviewer whose comments have helped us to improve the quality of the paper.