Structural and mutagenic analysis of the RM controller protein C.Esp1396I.
Structural and mutagenic analysis of the RM controller protein C.Esp1396I.
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DOI：
10.1371/journal.pone.0098365
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Kneale G
中科院分区：
文献类型：
--
作者：
Martin RN;McGeehan JE;Kneale G
细菌限制修饰（RM）系统由两种互补的酶活性组成，它们阻止细菌细胞中外源DNA的建立：DNA甲基化和DNA限制。这两种活性受到严格调控，以防止过度甲基化或自动限制。许多II型RM系统采用控制器（C）蛋白作为内切核酸酶基因（在某些情况下，甲基转移酶基因也是）的转录调节因子。迄今为止，所有解析的C-蛋白质/DNA-蛋白质复合物的高分辨率结构都与C.Esp1396I有关，从中可以观察到特定氨基酸残基与DNA碱基和/或磷酸骨架的相互作用。在这里，我们提出了C.Esp1396I的关键DNA结合残基的一系列突变的结构和DNA结合数据。我们的研究结果表明，突变的骨干结合残基（Y37，S52）有一个较小的影响，DNA结合亲和力比突变的那些残基直接结合的碱基（T36，R46），和贡献的每个侧链的结合能进行了比较。突变体和天然蛋白质的高分辨率X射线晶体结构表明，蛋白质的折叠不受突变的影响，但也揭示了与DNA序列识别相关的柔性环构象的变化。由于酪氨酸残基Y37有助于天然复合物中的DNA弯曲，我们已经通过X射线晶体学解决了Y37 F突变蛋白/DNA复合物的结构，使我们能够直接比较突变体和天然复合物中的DNA结构。
Bacterial restriction-modification (RM) systems are comprised of two complementary enzymatic activities that prevent the establishment of foreign DNA in a bacterial cell: DNA methylation and DNA restriction. These two activities are tightly regulated to prevent over-methylation or auto-restriction. Many Type II RM systems employ a controller (C) protein as a transcriptional regulator for the endonuclease gene (and in some cases, the methyltransferase gene also). All high-resolution structures of C-protein/DNA-protein complexes solved to date relate to C.Esp1396I, from which the interactions of specific amino acid residues with DNA bases and/or the phosphate backbone could be observed. Here we present both structural and DNA binding data for a series of mutations to the key DNA binding residues of C.Esp1396I. Our results indicate that mutations to the backbone binding residues (Y37, S52) had a lesser affect on DNA binding affinity than mutations to those residues that bind directly to the bases (T36, R46), and the contributions of each side chain to the binding energies are compared. High-resolution X-ray crystal structures of the mutant and native proteins showed that the fold of the proteins was unaffected by the mutations, but also revealed variation in the flexible loop conformations associated with DNA sequence recognition. Since the tyrosine residue Y37 contributes to DNA bending in the native complex, we have solved the structure of the Y37F mutant protein/DNA complex by X-ray crystallography to allow us to directly compare the structure of the DNA in the mutant and native complexes.
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DOI：
10.1107/s090744491003982x
发表时间：
2011-04
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Evans PR
通讯作者：
Evans PR
DOI：
10.1107/s0907444905036693
发表时间：
2006-01-01
影响因子：
2.2
作者：
Evans, P
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Evans, P
影响因子：
14.9
作者：
Lavery R;Moakher M;Maddocks JH;Petkeviciute D;Zakrzewska K
通讯作者：
Zakrzewska K
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
DOI：
10.1107/s0907444909042073
发表时间：
2010-01
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Chen VB;Arendall WB 3rd;Headd JJ;Keedy DA;Immormino RM;Kapral GJ;Murray LW;Richardson JS;Richardson DC
通讯作者：
Richardson DC