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

<div><p>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 (<i>N</i>) shows a nonmonotonic dependence on urea concentration indicating a unique multistep denaturation process, <i>N</i> → <i>I</i>₁<i>I</i>₂<i>U</i>, with two intermediate states <i>I</i>₁ and <i>I</i>₂. BoNT/A loses almost all its secondary structure in 3.75 M urea (<i>I</i>₁), yet it displays a native-like secondary structure in 5 M urea (<i>I</i>₂). 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 <i>I</i>₂, all the states revert back to full enzymatic activity for SNAP-25 including the unfolded state <i>U</i> 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.</p></div