Inference of relationships in the 'twilight zone' of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII.
Inference of relationships in the 'twilight zone' of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII.
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DOI：
10.1093/nar/gki213
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发表时间：
2005
影响因子：
14.9
通讯作者：
Skowronek KJ
中科院分区：
文献类型：
--
作者：
Pawlak SD;Radlinska M;Chmiel AA;Bujnicki JM;Skowronek KJ
到目前为止，使用传统方法鉴定II型限制性内切酶（REases）中功能重要残基一直很困难。即使已知的REase结构共享一个折叠和边缘可识别的活性位点，除非在识别相同或非常相似序列的蛋白质之间进行比较，否则总体序列相似性在统计上是微不足道的。Bsp6I是II型酶，它识别回文DNA序列5 ‘ gcngc，并在两条链上的胞嘧啶和未指定的核苷酸之间切割，产生具有5 ’突出的单核苷酸的双链断裂。目前还没有解出的酶结构能够识别相似的DNA靶标或产生具有相似特征的裂解产物。在直接比较中，Bsp6I序列显示与已知结构的酶没有显著的相似性。然而，使用折叠识别方法，我们已经确定了Bsp6I和pvii结构之间的远程关系。从Bsp6I与PvuII的序列结构比对出发，构建了Bsp6I的同源性模型，并利用该模型预测了Bsp6I的功能重要区域。同源性模型被预测对二聚化、DNA结合和催化重要的残基的定点突变所支持。在结构基因组学时代，完成蛋白质超家族中序列-结构-功能关系的图谱成为一项重要任务，我们的研究可以作为未来分析由很少或没有序列相似性的强烈分化成员组成的超家族的范例。
Thus far, identification of functionally important residues in Type II restriction endonucleases (REases) has been difficult using conventional methods. Even though known REase structures share a fold and marginally recognizable active site, the overall sequence similarities are statistically insignificant, unless compared among proteins that recognize identical or very similar sequences. Bsp6I is a Type II REase, which recognizes the palindromic DNA sequence 5′GCNGC and cleaves between the cytosine and the unspecified nucleotide in both strands, generating a double-strand break with 5′-protruding single nucleotides. There are no solved structures of REases that recognize similar DNA targets or generate cleavage products with similar characteristics. In straightforward comparisons, the Bsp6I sequence shows no significant similarity to REases with known structures. However, using a fold-recognition approach, we have identified a remote relationship between Bsp6I and the structure of PvuII. Starting from the sequence–structure alignment between Bsp6I and PvuII, we constructed a homology model of Bsp6I and used it to predict functionally significant regions in Bsp6I. The homology model was supported by site-directed mutagenesis of residues predicted to be important for dimerization, DNA binding and catalysis. Completing the picture of sequence–structure–function relationships in protein superfamilies becomes an essential task in the age of structural genomics and our study may serve as a paradigm for future analyses of superfamilies comprising strongly diverged members with little or no sequence similarity.
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影响因子：
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