Insights into the molecular recognition of the 5′-GNN-3′ family of DNA sequences by zinc finger domains
Insights into the molecular recognition of the 5′-GNN-3′ family of DNA sequences by zinc finger domains
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DOI：
10.1006/jmbi.2000.4133
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发表时间：
2000-11-03
影响因子：
5.6
通讯作者：
Barbas, CF
中科院分区：
文献类型：
--
作者：
Dreier, B;Segal, DJ;Barbas, CF
为了构建识别所有可能的64个DNA三元组的锌指域，有必要了解分子水平上蛋白质/DNA相互作用的机理。以前，我们报道了16个锌指结构域，这些结构域已详细介绍了与DNA序列的5'-GNN-3'家族特别结合。这些结构域构成的人工转录因子可以调节内源基因的表达。这些结构域是通过噬菌体选拔选择创建的，然后是定点诱变。总共分析了三域锌指蛋白的84个突变体的DNA结合特异性。在这里，我们报告了这项系统和广泛的诱变研究的结果。通过将特异性数据与计算机建模和比较与NMR和晶体学研究的已知结构数据相结合，获得了对锌指/DNA相互作用的新见解。该分析表明，这些蛋白质中的某些蛋白质中有异常的跨链和螺旋间接触，即使在侧翼锌指域约束时，识别螺旋对DNA的一般取向也是柔性的。这些发现不利于现有简单识别代码的实用性，并表明在大多数情况下，不能单独从噬菌体显示中获得高度特定的域，而仅与理性设计结合使用。锌指/DNA相互作用的分子基础很复杂，其理解取决于大量蛋白质的分析。这种理解应该使我们能够迅速完善其他锌指结构域的特异性，以及用这些结构域构建的聚二酰基蛋白来识别扩展的DNA序列。 （c）2000学术出版社。
In order to construct zinc finger domains that recognize all of the possible 64 DNA triplets, it is necessary to understand the mechanisms of protein/DNA interactions on the molecular level. Previously we reported 16 zinc finger domains which had been characterized in detail to bind specifically to the 5'-GNN-3' family of DNA sequences. Artificial transcription factors constructed from these domains can regulate the expression of endogenous genes. These domains were created by phage-display selection followed by site-directed mutagenesis. A total of 84 mutants of a three-domain zinc finger protein have been analyzed for their DNA-binding specificity. Here, we report the results of this systematic and extensive mutagenesis study. New insights into zinc finger/DNA interactions were obtained by combining specificity data with computer modeling and comparison with known structural data from NMR and crystallographic studies. This analysis suggests that unusual cross-strand and inter-helical contacts are made by some of these proteins, and the general orientation of the recognition helix to the DNA is flexible, even when constrained by flanking zinc finger domains. These findings disfavor the utility of existing simple recognition codes and suggest that highly specific domains cannot be obtained from phage display alone in most cases, but only in combination with rational design. The molecular basis of zinc finger/DNA interaction is complex and its understanding is dependent on the analysis of a large number of proteins. This understanding should enable us to refine rapidly the specificity of other zinc finger domains, as well as polydactyl proteins constructed with these domains to recognize extended DNA sequences. (C) 2000 Academic Press.