Guanine binding site of the Nicotiana glutinosa ribonuclease NW revealed by X-ray crystallography
Guanine binding site of the Nicotiana glutinosa ribonuclease NW revealed by X-ray crystallography
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DOI：
10.1021/bi026247l
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发表时间：
2002-12-24
期刊：
影响因子：
2.9
通讯作者：
Kimura, M
中科院分区：
文献类型：
--
作者：
Kawano, S;Kakuta, Y;Kimura, M
核糖核酸酶NW（RNase NW）是烟草叶片中的创伤诱导型RNase，优先切割鸟苷酸。我们使用表达载体pPIC 9 K在甲基营养型酵母Pichia pastoris中表达编码RNase NW的cDNA，并且使用柱层析将分泌到培养基中的所得重组RNase NW（ryRNaseNW）纯化至表观均一性。通过用番茄RNA酶LE作为搜索模型进行分子置换，在1.5埃分辨率下确定与5 ′-GMP结合的ryRNA酶NW的晶体结构。RNase NW在结构上属于（α + β）类蛋白质，具有八个螺旋（五个α-螺旋和三个3（10）螺旋）和六个β-链，其结构与其他植物RNase的结构高度相似，包括来自苦瓜种子的尿苷酸优先RNase MC 1。5 '-GMP的鸟嘌呤环位于由Tyr 17、Tyr 71、Ala 80、Leu 79和Phe 89组成的分子表面的疏水口袋中：鸟嘌呤碱基夹在Tyr 17和Phe 89的芳香族侧链之间。此外，鸟嘌呤碱基通过Gln 12和Thr 78的侧链以及Leu 79的主链的氢键网络而牢固地稳定。因此，Gln 12，Tyr 17，Thr 78，Leu 79和Phe 89负责通过RNase NW识别鸟嘌呤碱基，这些发现提供了对RNase NW优先切割鸟苷酸的方式的深入了解。
Ribonuclease NW (RNase NW), the wound-inducible RNase in Nicotiana glutinosa leaves, preferentially cleaves guanylic acid. We expressed the cDNA encoding RNase NW in the methylotrophic yeast Pichia pastoris using the expression vector pPIC9K, and the resulting recombinant RNase NW (ryRNaseNW) secreted into medium was purified to apparent homogeneity using column chromatography. The crystal structure of ryRNase NW bound to 5'-GMP was determined at 1.5 Angstrom resolution by molecular replacement with tomato RNase LE as a search model. The RNase NW structurally belongs to the (alpha + beta) class of proteins, having eight helices (five alpha-helices and three 3(10) helices) and six beta-strands, and its structure is highly similar to those of other plant RNases, including a uridylic acid preferential RNase MC1 from bitter gourd seeds. The guanine ring of 5'-GMP lies in a hydrophobic pocket of the molecular surface composed of Tyr17, Tyr71, Ala80, Leu79, and Phe89: the guanine base is sandwiched between aromatic side chains of Tyr17 and Phe89. In addition, the guanine base is firmly stabilized by a network of hydrogen bonds of the side chains of Gln12 and Thr78, as well as of the main chain of Leu79. Therefore, Gln12, Tyr17, Thr78, Leu79, and Phe89 are responsible for recognition of the guanine base by RNase NW, findings which provide insight into the manner in which RNase NW preferentially cleaves guanylic acid.