Novel protein fold discovered in the PabI family of restriction enzymes.
Novel protein fold discovered in the PabI family of restriction enzymes.
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DOI：
10.1093/nar/gkm091
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发表时间：
2007
影响因子：
14.9
通讯作者：
Kobayashi, Ichizo
中科院分区：
文献类型：
--
作者：
Miyazono, Ken-ichi;Watanabe, Miki;Kosinski, Jan;Ishikawa, Ken;Kamo, Masayuki;Sawasaki, Tatsuya;Nagata, Koji;Bujnicki, Janusz M;Endo, Yaeta;Tanokura, Masaru;Kobayashi, Ichizo
虽然许多DNA结合蛋白的结构已经被解决，但它们落入有限数量的折叠中。在这里，我们描述了一种方法，导致发现一种新的DNA结合折叠。基于II型限制-修饰基因复合物作为移动的元件的行为，我们的早期工作通过比较密切相关的基因组，在深海热球菌中鉴定了限制酶R.PabI及其同源修饰酶。虽然修饰甲基转移酶很容易识别，但R.PabI被预测具有新的3D结构。我们表达细胞毒性R.PabI在小麦胚芽为基础的无细胞翻译系统，并确定其晶体结构。R.PabI采用了一种新的蛋白质折叠。同型二聚体R.PabI具有弯曲的反平行β-折叠，形成“半管”。突变和计算机DNA结合分析已将其指定为双链DNA结合位点。与分析的大多数限制性内切酶不同，R.PabI能够在不存在Mg 2+的情况下切割DNA。这些结果证明了基因组比较和基于小麦胚芽的系统在发现移动的DNA酶中的新DNA结合基序以及在水平转移基因中的新蛋白质折叠中的价值。
Although structures of many DNA-binding proteins have been solved, they fall into a limited number of folds. Here, we describe an approach that led to the finding of a novel DNA-binding fold. Based on the behavior of Type II restriction–modification gene complexes as mobile elements, our earlier work identified a restriction enzyme, R.PabI, and its cognate modification enzyme in Pyrococcus abyssi through comparison of closely related genomes. While the modification methyltransferase was easily recognized, R.PabI was predicted to have a novel 3D structure. We expressed cytotoxic R.PabI in a wheat-germ-based cell-free translation system and determined its crystal structure. R.PabI turned out to adopt a novel protein fold. Homodimeric R.PabI has a curved anti-parallel β-sheet that forms a ‘half pipe’. Mutational and in silico DNA-binding analyses have assigned it as the double-strand DNA-binding site. Unlike most restriction enzymes analyzed, R.PabI is able to cleave DNA in the absence of Mg2+. These results demonstrate the value of genome comparison and the wheat-germ-based system in finding a novel DNA-binding motif in mobile DNases and, in general, a novel protein fold in horizontally transferred genes.