Nickel(ii)-promoted specific hydrolysis of zinc finger proteins
Nickel(ii)-promoted specific hydrolysis of zinc finger proteins
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DOI：
10.1039/c8mt00098k
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发表时间：
2018-08-01
期刊：
影响因子：
3.4
通讯作者：
Bal, Wojciech
中科院分区：
文献类型：
--
作者：
Belczyk-Ciesielska, Agnieszka;Csipak, Brigitta;Bal, Wojciech
在这项工作中，我们证明前面描述的序列特异性 Ni(ii) 依赖性水解肽键裂解反应可以在复杂的金属蛋白分子中进行，例如 Cys(2)His(2) 锌指蛋白。具有 (Ser/Thr)-X-His 序列的锌指单元内的裂解不受 Zn(ii) 离子的存在阻碍。它会导致 Zn(ii) 离子的损失、SH 基团的氧化，从而导致功能结构的崩溃。我们表明，锌指结构域中的此类天然 Ni(ii) 切割位点可以被编辑掉，而不会影响 DNA 结合特异性。在编辑的锌指和亲和标签之间插入 Ni(ii) 敏感序列，可以在蛋白质纯化后通过 Ni(ii) 离子去除后者序列。我们已经证明，即使存在结合 N 端 His 标签的金属离子，也可以执行该反应。裂解产物保持了涉及 Zn(ii) 离子的天然锌指结构。质谱显示 Ni(ii) 离子通过 N 末端 (Ser/Thr)-X-His 三肽片段与水解蛋白质产物保持配位。 Ni(ii) 依赖性蛋白质水解受 Ni(ii) 浓度、pH 和反应温度的影响，这一事实为新型调控 DNA 效应器设计提供了平台。
In this work we demonstrate that the previously described reaction of sequence specific Ni(ii)-dependent hydrolytic peptide bond cleavage can be performed in complex metalloprotein molecules, such as the Cys(2)His(2) zinc finger proteins. The cleavage within a zinc finger unit possessing a (Ser/Thr)-X-His sequence is not hindered by the presence of the Zn(ii) ions. It results in loss of the Zn(ii) ion, oxidation of the SH groups and thus, in a collapse of the functional structure. We show that such natural Ni(ii)-cleavage sites in zinc finger domains can be edited out without compromising the DNA binding specificity. Inserting a Ni(ii)-susceptible sequence between the edited zinc finger and an affinity tag allows for removal of the latter sequence by Ni(ii) ions after the protein purification. We have shown that this reaction can be executed even when a metal ion binding N-terminal His-tag is present. The cleavage product maintains the native zinc finger structure involving Zn(ii) ions. Mass spectra revealed that a Ni(ii) ion remains coordinated to the hydrolyzed protein product through the N-terminal (Ser/Thr)-X-His tripeptide segment. The fact that the Ni(ii)-dependent protein hydrolysis is influenced by the Ni(ii) concentration, pH and temperature of the reaction provides a platform for novel regulated DNA effector design.