大肠杆菌YacG锌指结构的金属结合及功能特性
发布时间：2018-10-08 20:57
【摘要】：Yac G蛋白是一种能够抑制大肠杆菌促旋酶(E.coli gyrase)活性的内源性小分子蛋白质,仅由65个氨基酸残基组成。核磁共振(NMR)研究发现,Yac G结构中含有1个Cys-X2-Cys-X15-CysX3-Cys序列的锌指结构域,然而其作用并不清楚。本研究发现,在添加外源锌或者铁的M9基础培养基中,表达并纯化得到分别含有锌和铁的Yac G蛋白,而在同时添加铁和L-半胱氨酸的M9基础培养基中可以纯化得到含有铁硫簇的蛋白质。这表明,Yac G不仅是一个锌指蛋白,也是铁结合或铁硫簇结合蛋白。定点突变实验发现,Yac G锌指结构中的4个半胱氨酸残基突变后,其结合的锌、铁、铁硫簇的含量都显著下降。这提示,锌结合、铁结合以及铁硫簇结合的位点均位于锌指结构域中的4个半胱氨酸残基。体内Yac G过表达实验显示,用IPTG在大肠杆菌体内诱导表达野生型Yac G蛋白会导致其生长明显受到抑制,而过表达突变体蛋白(Yac G-C12/28S)对其生长的抑制作用将会减弱。体外实验进一步发现,锌结合、铁结合以及铁硫簇结合形式的Yac G蛋白对E.coli gyrase促DNA螺旋活性的抑制作用没有明显差别,但是锌指结构突变体蛋白(Yac G-C12/28S)对gyrase活性的抑制作用显著减弱。这说明,完整的锌指结构对Yac G抑制gyrase活性的功能具有重要作用。此研究有可能为gyrase抑制剂类抗生素药物的研发提供有用的线索。
[Abstract]:Yac G protein is an endogenous small molecule protein which can inhibit the activity of Escherichia coli (E.coli gyrase) and consists of only 65 amino acid residues. A zinc finger domain with Cys-X2-Cys-X15-CysX3-Cys sequence was found in the structure of Yac G by nuclear magnetic resonance (NMR), but its role is not clear. In this study, we found that Yac G protein containing zinc and iron was expressed and purified in M9 basal medium supplemented with exogenous zinc or iron, respectively. The protein containing iron and sulfur clusters could be purified from M9 basal medium supplemented with iron and L-cysteine. This indicates that Yac G is not only a zinc finger protein, but also an iron-bound or iron-sulfur cluster binding protein. The results of site-directed mutation showed that the contents of bound zinc and iron-sulfur clusters decreased significantly after four cysteine residues of Yac G zinc finger structure were mutated. The results suggested that the sites of zinc binding, iron binding and iron-sulfur cluster binding were all located at four cysteine residues in the zinc-finger domain. Overexpression of Yac G in vivo showed that the expression of wild-type Yac G protein induced by IPTG in Escherichia coli resulted in obvious inhibition of its growth, but the inhibitory effect of overexpression mutant protein (Yac G-C12/28S) on its growth was weakened. It was further found that there was no significant difference in the inhibitory effects of zinc binding, iron binding and iron-sulfur cluster binding Yac G protein on the DNA helical activity induced by E.coli gyrase in vitro. However, zinc finger structural mutant protein (Yac G-C12/28S) inhibited the activity of gyrase significantly. This suggests that the intact zinc finger structure plays an important role in the inhibition of gyrase activity by Yac G. This study may provide useful clues for the development of gyrase inhibitor antibiotics.
【作者单位】： 温州医科大学检验医学院生命科学学院检验医学教育部重点实验室;温州市中心医院风湿免疫科;
【基金】：国家自然科学基金项目(No.31100576) 浙江省公益技术研究项目(No.2016C33027)资助~~
【分类号】：R378
本文编号：2258235
[Abstract]:Yac G protein is an endogenous small molecule protein which can inhibit the activity of Escherichia coli (E.coli gyrase) and consists of only 65 amino acid residues. A zinc finger domain with Cys-X2-Cys-X15-CysX3-Cys sequence was found in the structure of Yac G by nuclear magnetic resonance (NMR), but its role is not clear. In this study, we found that Yac G protein containing zinc and iron was expressed and purified in M9 basal medium supplemented with exogenous zinc or iron, respectively. The protein containing iron and sulfur clusters could be purified from M9 basal medium supplemented with iron and L-cysteine. This indicates that Yac G is not only a zinc finger protein, but also an iron-bound or iron-sulfur cluster binding protein. The results of site-directed mutation showed that the contents of bound zinc and iron-sulfur clusters decreased significantly after four cysteine residues of Yac G zinc finger structure were mutated. The results suggested that the sites of zinc binding, iron binding and iron-sulfur cluster binding were all located at four cysteine residues in the zinc-finger domain. Overexpression of Yac G in vivo showed that the expression of wild-type Yac G protein induced by IPTG in Escherichia coli resulted in obvious inhibition of its growth, but the inhibitory effect of overexpression mutant protein (Yac G-C12/28S) on its growth was weakened. It was further found that there was no significant difference in the inhibitory effects of zinc binding, iron binding and iron-sulfur cluster binding Yac G protein on the DNA helical activity induced by E.coli gyrase in vitro. However, zinc finger structural mutant protein (Yac G-C12/28S) inhibited the activity of gyrase significantly. This suggests that the intact zinc finger structure plays an important role in the inhibition of gyrase activity by Yac G. This study may provide useful clues for the development of gyrase inhibitor antibiotics.
【作者单位】： 温州医科大学检验医学院生命科学学院检验医学教育部重点实验室;温州市中心医院风湿免疫科;
【基金】：国家自然科学基金项目(No.31100576) 浙江省公益技术研究项目(No.2016C33027)资助~~
【分类号】：R378
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1 白毅;我国锌指抗病毒蛋白研究获进展[N];中国医药报;2008年
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