The sulfur atom of phosphorothioated DNA (PT-DNA) is coordinated by a surface cavity in the conserved sulfur-binding domain (SBD) of type IV restriction enzymes. However, some SBDs cannot recognize the sulfur atom in some sequence contexts. To illustrate the structural determinants for sequence specificity, we resolved the structure of SBD_Spr, from endonuclease SprMcrA, in complex with DNA of G_PSGCC, G_PSATC and G_PSAAC contexts. Structural and computational analyses explained why it binds the above PT-DNAs with an affinity in a decreasing order. The structural analysis of SBD_Spr–G_PSGCC and SBD_Sco–G_PSGCC, the latter only recognizes DNA of G_PSGCC, revealed that a positively charged loop above the sulfur-coordination cavity electrostatically interacts with the neighboring DNA phosphate linkage. The structural analysis indicated that the DNA–protein hydrogen bonding pattern and weak non-bonded interaction played important roles in sequence specificity of SBD protein. Exchanges of the positively-charged amino acid residues with the negatively-charged residues in the loop would enable SBD_Sco to extend recognization for more PT-DNA sequences, implying that type IV endonucleases can be engineered to recognize PT-DNA in novel target sequences.

中文翻译：

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DNA主链相互作用影响DNA硫结合域的序列特异性：结构分析的启示。

硫代磷酸化的DNA（PT-DNA）的硫原子由IV型限制酶的保守硫结合域（SBD）中的表面空腔配位。但是，某些SBD在某些序列环境中无法识别硫原子。为了说明序列特异性的结构决定因素，我们从核酸内切酶SprMcrA中解析了SBD _Spr的结构，并与G _PS GCC，G _PS ATC和G _PS AAC环境的DNA进行了复合。结构和计算分析解释了为什么它以递减的顺序结合上述PT-DNA。SBD _Spr –G _PS GCC和SBD _Sco –G _PS的结构分析GCC，后者仅识别G _PS GCC的DNA ，揭示了硫配位腔上方的带正电荷的环与邻近的DNA磷酸键静电相互作用。结构分析表明，DNA-蛋白质的氢键模式和弱的非键相互作用在SBD蛋白质的序列特异性中起重要作用。环中带正电荷的氨基酸残基与带负电荷的残基的交换将使SBD _Sco扩展对更多PT-DNA序列的识别，这意味着可以对IV型核酸内切酶进行改造以识别新的靶序列中的PT-DNA。