Role of Zinc in the Structure and Toxic Activity of Botulinum Neurotoxin

Abstract

Zn2+-protease activity of botulinum neurotoxin causes the blockage of neurotransmitter release resulting in botulism disease. We have investigated the role of Zn2+ in the biological activity of type A botulinum neurotoxin by removing the bound Zn2+ by EDTA treatment, followed by monitoring its structure in terms of secondary and tertiary folding (second derivative UV, FT-IR, and circular dichroism spectroscopy) and function in terms of its effect on the release of norepinephrine from PC12 cells. The single Zn2+ bound to each neurotoxin molecule was reversibly removed by EDTA treatment, whereas the biological activity of the neurotoxin was irreversibly lost. Based on the Amide III IR spectral analysis, the alpha-helical content of neurotoxin increased from 29% to 42% upon removal of Zn2+, which reverted to 31% upon treatment with 1:5 molar excess of exogenous Zn2+. Second derivative UV spectroscopy revealed no change in surface topography of Tyr residues with removal of Zn2+. However, near-UV circular dichroism signals suggested significant alterations in the topography of Phe and Tyr residues that could be buried in the protein matrix. Thermal unfolding experiments suggested that removal of Zn2+ results in the formation of the molten globule-like structure of type A botulinum neurotoxin. Tertiary structural changes introduced by Zn2+ removal were irreversible, which correlated well with the irreversibility of the biological activity of the neurotoxin. On the basis of these results, we suggest that Zn2+ plays a significant structural role in addition to its catalytic role in Zn2+-protease activity of type A botulinum neurotoxin.

Comparison of Extracellular and Intracellular Potency of Botulinum Neurotoxins

“…In contrast to the EDTA effect, the Zn 2ϩ -specific chelator N,N,NЈ,NЈ-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) reversibly inhibits the BoNTs in both assays. The results presented here rectify an apparent contradiction between previous reports in which separate studies examined either EDTA or TPEN, leading to opposite results and different interpretations (15,27,33,46). The current data allow the coexistence of the competing observations and suggest that the discrepancies were due to different model systems and different reaction conditions employed in each study.…”

“…In contrast, the BoNT/E LC required longer incubation with TPEN-at least 60 min to achieve total inhibition. Therefore, extracellular BoNT/E seemed EDTA has previously been shown to physically denature the tertiary structure of BoNT/A, which was attributed to removal of Zn 2ϩ (15,27). When 65 Zn 2ϩ was used, the binding was shown to be reversible but the conformational changes to the LC were not corrected upon reacquisition of 65 Zn 2ϩ .…”

“…This was important since previous studies had concluded that Zn 2ϩ chelation destroys BoNT/A biological function (15). Each BoNT (0.5 M) was treated with TPEN for 90 min at room temperature prior to being diluted into culture medium (250 pM final toxin, 50 nM 10 M TPEN, 1.5 M Zn 2ϩ ).…”

The Role of Zinc Binding in the Biological Activity of Botulinum Toxin

“…This involved the use of soluble and immobilized chelators. One of the soluble chelators, EDTA, was the same as that used by Fu et al (9), but the results were profoundly different. In the hands of the present investigators, both EDTA and TPEN caused virtually complete loss of enzyme activity as measured by the cleavage of recombinant substrate.…”

“…More recently, Fu et al (9) have published findings that seem to contradict those just described. They found that the use of a chelator to remove Zn 2ϩ from toxin led to irreversible changes in tertiary structure as measured by various light-scattering techniques.…”