Identification of a zinc finger domain in the human NEIL2 (Nei-like-2) protein
Identification of a zinc finger domain in the human NEIL2 (Nei-like-2) protein
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DOI：
10.1074/jbc.m406224200
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发表时间：
2004-11-05
影响因子：
4.8
通讯作者：
Hazra, TK
中科院分区：
文献类型：
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作者：
Das, A;Rajagopalan, L;Hazra, TK
NEIL 2（Nei-like-2）蛋白是一种DNA糖基化酶/AP裂解酶，与大肠杆菌的Nei和Fpg具有相同的结构特征和反应机制。氨基酸序列分析表明NEIL 2具有锌指样Nei/Fpg结构。然而，存在于NEIL 2的C末端附近的Cys-X-2-His-X-16-Cys-X-2-Cys（CHCC）基序不同于C4型的Nei/Fpg的锌指基序。在这里，我们通过电感耦合等离子体质谱法显示了NEIL 2中等摩尔量的锌的存在。Cys-291、His-295、Cys-315和Cys-318的个别突变（用于配位锌的候选残基）通过废除其DNA结合活性而使酶失活。H295 A和C318 S突变体也显示缺乏结合锌，并且通过CD光谱分析揭示了它们的二级结构的显著变化。分子模拟显示NEIL 2的Arg-310是其锌结合口袋中的关键残基，其在整个Fpg/Nei家族中高度保守。R310 Q突变显著降低了NEIL 2的活性。因此，我们得出结论，在NEIL 2的锌指基序是必不可少的结构完整性和酶活性。
The recently identified human NEIL2 (Nei-like-2) protein, a DNA glycosylase/AP lyase specific for oxidatively damaged bases, shares structural features and reaction mechanism with the Escherichia coli DNA glycosylases, Nei and Fpg. Amino acid sequence analysis of NEIL2 suggested it to have a zinc finger-like Nei/Fpg. However, the Cys-X-2-His-X-16-Cys-X-2-Cys (CHCC) motif present near the C terminus of NEIL2 is distinct from the zinc finger motifs of Nei/Fpg, which are of the C4 type. Here we show the presence of an equimolar amount of zinc in NEIL2 by inductively coupled plasma mass spectrometry. Individual mutations of Cys-291, His-295, Cys-315, and Cys-318, candidate residues for coordinating zinc, inactivated the enzyme by abolishing its DNA binding activity. H295A and C318S mutants were also shown to lack bound zinc, and a significant change in their secondary structure was revealed by CD spectra analysis. Molecular modeling revealed Arg-310 of NEIL2 to be a critical residue in its zinc binding pocket, which is highly conserved throughout the Fpg/Nei family. A R310Q mutation significantly reduced the activity of NEIL2. We thereby conclude that the zinc finger motif in NEIL2 is essential for its structural integrity and enzyme activity.