A homology model of restriction endonuclease SfiI in complex with DNA
A homology model of restriction endonuclease SfiI in complex with DNA
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DOI：
10.1186/1472-6807-5-2
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发表时间：
2005-01-24
影响因子：
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通讯作者：
Skowronek, KJ
中科院分区：
文献类型：
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作者：
Chmiel, AA;Bujnicki, JM;Skowronek, KJ
背景：限制性内切酶（REases）是重组DNA技术中常用的商业试剂。它们是研究蛋白质-DNA相互作用的有吸引力的模型和蛋白质工程的有价值的靶标。然而，它们是非常不同的：一个典型的REase的氨基酸序列通常没有显示出与任何其他蛋白质的可检测的相似性，与其他REase识别相同或非常相似的序列的罕见例外。从结构分析和生物信息学研究中，已经了解到一些REase属于至少四个不相关且结构不同的核酸酶超家族，PD-DxK、PLD、HNH和GIY-YIG。因此，它们是结构预测和基于同源性的序列-功能关系推断的非常困难的目标，并且绝大多数REase在结构和进化上仍然未分类。结果：Sfil是一种REase，其识别中断的回文序列5 'GGCCNNNGGCC 3'，并且在切割时产生3 nt长的3 '突出端。Sfil是原型IIF型酶，其作为四聚体起作用并且以协同方式切割两个拷贝的识别位点。它的序列与其他蛋白质没有相似性，并且对其活性位点或对寡聚化重要的残基的定位一无所知。使用用于蛋白质折叠识别的穿线方法，我们鉴定了Sfil和Bgll之间的远程关系，Bgll是来自核酸酶的PD-DxK超家族的二聚体IIP型限制酶，其识别5 'GCCNNNNGGC 3'序列并且其与底物DNA复合的结构是可用的。我们构建了一个同源性模型的SfiI在复杂的目标序列，并用它来预测残基的二聚体，四聚体，DNA结合和catalyst.Conclusions重要：生物信息学分析表明，Sfil，一个类型IIF酶，是更密切相关的Bgll，一个“正统”类型IIP限制性内切酶，比任何其他REase，包括其他类型IIF REases与已知的结构，如NgoMIV。NgoMIV和BglI分别属于PD-DxK超家族的两个不同的、非常遥远相关的分支：α类（EcoRV样）和β类（EcoRV样）。因此，我们的分析提供的证据表明，在PD-DxK超家族的REases的进化中，以协调一致的方式四聚化和切割两个DNA序列的能力独立地发展了至少两次。Sfil模型也将作为进一步实验分析的方便平台。
Background: Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified.Results: Sfil is a REase which recognizes the interrupted palindromic sequence 5 ' GGCCNNNNNGGCC3' and generates 3 nt long 3 ' overhangs upon cleavage. Sfil is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between Sfil and Bgll, a dimeric Type IIP restriction enzyme from the PD- DxK superfamily of nucleases, which recognizes the 5 ' GCCNNNNNGGC3 ' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis.Conclusions: The bioinformatics analysis suggest that Sfil, a Type IIF enzyme, is more closely related to Bgll, an "orthodox" Type IIP restriction enzyme, than to any other REase, including other Type IIF REases with known structures, such as NgoMIV. NgoMIV and BglI belong to two different, very remotely related branches of the PD-DxK superfamily: the alpha-class (EcoRV- like), and the beta-class (EcoRV-like), respectively. Thus, our analysis provides evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily of REases. The model of Sfil will also serve as a convenient platform for further experimental analyses.