Iron inhibits Escherichia coli topoisomerase I activity by targeting the first two zinc‐binding sites in the C‐terminal domain
Iron inhibits Escherichia coli topoisomerase I activity by targeting the first two zinc‐binding sites in the C‐terminal domain
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DOI：
10.1002/pro.2542
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发表时间：
2014-11
期刊：
影响因子：
8
通讯作者：
Wu Wang;Xiaolu Su;Xiaobing Wang;Juanjuan Yang;Ting Zhang;Mao-feng Wang;Rugen Wan;Guoqiang Tan;Jianxin Lu
中科院分区：
文献类型：
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作者：
Wu Wang;Xiaolu Su;Xiaobing Wang;Juanjuan Yang;Ting Zhang;Mao-feng Wang;Rugen Wan;Guoqiang Tan;Jianxin Lu
大肠杆菌DNA拓扑异构酶I （TopA）含有一个67 kDa的N端催化结构域和一个30 kDa的C端锌结合区域（ZD结构域），ZD结构域具有三个相邻的四半胱氨酸锌结合基元。先前的研究表明，大肠杆菌TopA可以结合铁和锌，而铁在TopA中的结合导致无法解开负超卷曲的DNA。在这里，我们报道了每个大肠杆菌TopA单体通过ZD结构域的前两个锌结合基序结合一个铁原子，并且第一和第二个锌结合基序都是TopA中铁结合所必需的。位点定向诱变研究进一步表明，虽然第三个锌结合基序的突变对TopA的活性影响很小，但TopA中前两个锌结合基序的突变极大地降低了TopA在体内和体外的拓扑异构酶活性，这表明TopA中前两个锌结合基序对其功能至关重要。DNA结合活性测定和固有色氨酸荧光测量表明，铁与TopA的结合可能降低ZD结构域的单链DNA结合活性，并改变TopA的蛋白质结构，从而调节拓扑异构酶的活性。
Escherichia coli DNA topoisomerase I (TopA) contains a 67 kDa N‐terminal catalytic domain and a 30 kDa C‐terminal zinc‐binding region (ZD domain) which has three adjacent tetra‐cysteine zinc‐binding motifs. Previous studies have shown that E. coli TopA can bind both iron and zinc, and that iron binding in TopA results in failure to unwind the negatively supercoiled DNA. Here, we report that each E. coli TopA monomer binds one atom of iron via the first two zinc‐binding motifs in ZD domain and both the first and second zinc‐binding motifs are required for iron binding in TopA. The site‐directed mutagenesis studies further reveal that while the mutation of the third zinc‐binding motif has very little effect on TopA's activity, mutation of the first two zinc‐binding motifs in TopA greatly diminishes the topoisomerase activity in vitro and in vivo, indicating that the first two zinc‐binding motifs in TopA are crucial for its function. The DNA‐binding activity assay and intrinsic tryptophan fluorescence measurements show that iron binding in TopA may decrease the single‐stranded (ss) DNA‐binding activity of ZD domain and also change the protein structure of TopA, which subsequently modulate topoisomerase activity.