Thermal stabilization of the catalytic domain of botulinum neurotoxin E by phosphorylation of a single tyrosine residue
Thermal stabilization of the catalytic domain of botulinum neurotoxin E by phosphorylation of a single tyrosine residue
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DOI：
10.1021/bi001919y
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发表时间：
2001-02-20
期刊：
影响因子：
2.9
通讯作者：
Ferrer-Montiel, A
中科院分区：
文献类型：
--
作者：
Blanes-Mira, C;Ibañez, C;Ferrer-Montiel, A
梭菌神经毒素的催化结构域是酪氨酸特异性蛋白激酶的底物。酪氨酸磷酸化的功能作用及其磷酸化位点的数量和位置仍然是难以捉摸的。我们已经使用重组肉毒杆菌神经毒素E（BoNT E）的催化结构域来研究这些问题。细菌表达和纯化的BoNT E催化结构域是完全活性的，并在体外被酪氨酸特异性激酶Src磷酸化。酪氨酸磷酸化的催化结构域增加了蛋白质的热稳定性，而不影响其蛋白水解活性。内肽酶的共价修饰促进了无序到有序的转变，如α-螺旋含量增加35%所证明的，这导致其变性温度增加4 ℃。在位置67处的酪氨酸的定点替换完全废除Src的磷酸盐掺入。组成性非磷酸化内肽酶突变体表现出的功能特性几乎相同的nonphosphorylated野生型催化结构域所显示的。这些发现表明梭菌神经毒素的催化结构域中存在单个磷酸化位点，并且其共价修饰主要调节蛋白质的热稳定性。
The catalytic domain of clostridial neurotoxins is a substrate of tyrosine-specific protein kinases. The functional role of tyrosine phosphorylation and also the number and location of its (their) phosphorylation site(s) are yet elusive. We have used the recombinant catalytic domain of botulinum neurotoxin E (BoNT E) to examine these issues. Bacterially expressed and purified BoNT E catalytic domain was fully active, and was phosphorylated in vitro by the tyrosine-specific kinase Src. Tyrosine phosphorylation of the catalytic domain increased the protein thermal stability without affecting its proteolytic activity. Covalent modification of the endopeptidase promoted a disorder-to-order transition, as evidenced by the 35% increment of the alpha -helical content, which resulted in a 4 degreesC increase of its denaturation temperature. Site-directed replacement of tyrosine at position 67 completely abolished phosphate incorporation by Src. Constitutively unphosphorylated endopeptidase mutants exhibited functional properties virtually identical to those displayed by the nonphosphorylated wild-type catalytic domain. These findings indicate the presence of a single phosphorylation site in the catalytic domain of clostridial neurotoxins, and that its covalent modification primarily modulates the protein thermostability.