Role of zinc in the structure and toxic activity of botulinum neurotoxin.
Role of zinc in the structure and toxic activity of botulinum neurotoxin.
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锌在肉毒杆菌神经毒素的结构和毒性活性中的作用。
DOI：
10.1021/bi9723966
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Singh,BR
中科院分区：
文献类型：
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作者：
Fu,FN;Lomneth,RB;Cai,S;Singh,BR
肉毒杆菌神经毒素的锌蛋白水解酶活性导致神经递质释放受阻，从而导致肉毒杆菌中毒。我们研究了锌离子在A型肉毒杆菌神经毒素生物活性中的作用，通过EDTA处理去除结合的锌离子，然后监测其二级和三级折叠的结构(二阶导数UV，FT-IR和圆二色谱)以及它对PC12细胞释放去甲肾上腺素的影响。EDTA处理可以可逆地去除与神经毒素分子结合的单一锌离子，而神经毒素的生物活性则不可逆转地丧失。根据酰胺Ⅲ红外光谱分析，神经毒素的α螺旋含量在去除锌离子后从29%增加到42%，而在外源锌离子摩尔比为1：5的情况下，又恢复到31%。二阶导数紫外可见光谱表明酪氨酸残基的表面形貌没有随着锌离子的去除而改变。然而，近紫外圆二色谱信号表明，Phe和Tyr残基的形貌发生了显著变化，这些变化可能隐藏在蛋白质基质中。热展开实验表明，锌离子的去除导致了A型肉毒神经毒素熔融球状结构的形成。锌离子去除引起的三级结构变化是不可逆的，这与神经毒素生物活性的不可逆性有很好的相关性。在这些结果的基础上，我们认为，除了对A型肉毒神经毒素的锌蛋白水解酶活性的催化作用外，锌离子还起着重要的结构作用。
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 α-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.