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Crystal structures of the RNA triphosphatase from Trypanosoma cruzi provide insights into how it recognizes the 5'-end of the RNA substrate.
Journal of Biological Chemistry ( IF 5.5 ) Pub Date : 2020-07-03 , DOI: 10.1074/jbc.ra119.011811
Yuko Takagi 1 , Naoyuki Kuwabara 2 , Truong Tat Dang 3 , Koji Furukawa 1 , C Kiong Ho 3
Affiliation  

RNA triphosphatase catalyzes the first step in mRNA cap formation, hydrolysis of the terminal phosphate from the nascent mRNA transcript. The RNA triphosphatase from the protozoan parasite Trypanosoma cruzi, TcCet1, belongs to the family of triphosphate tunnel metalloenzymes (TTMs). TcCet1 is a promising antiprotozoal drug target because the mechanism and structure of the protozoan RNA triphosphatases are completely different from those of the RNA triphosphatases found in mammalian and arthropod hosts. Here, we report several crystal structures of the catalytically active form of TcCet1 complexed with a divalent cation and an inorganic tripolyphosphate in the active-site tunnel at 2.20–2.51 Å resolutions. The structures revealed that the overall structure, the architecture of the tunnel, and the arrangement of the metal-binding site in TcCet1 are similar to those in other TTM proteins. On the basis of the position of three sulfate ions that cocrystallized on the positively charged surface of the protein and results obtained from mutational analysis, we identified an RNA-binding site in TcCet1. We conclude that the 5′-end of the triphosphate RNA substrate enters the active-site tunnel directionally. The structural information reported here provides valuable insight into designing inhibitors that could specifically block the entry of the triphosphate RNA substrate into the TTM-type RNA triphosphatases of T. cruzi and related pathogens.

中文翻译:

克氏锥虫 RNA 三磷酸酶的晶体结构提供了关于它如何识别 RNA 底物 5' 端的见解。

RNA 三磷酸酶催化 mRNA 帽形成的第一步,即新生 mRNA 转录物中末端磷酸盐的水解。来自原生动物寄生虫克氏锥虫的 RNA 三磷酸酶 TcCet1 属于三磷酸隧道金属酶 (TTM) 家族。TcCet1是一个有前途的抗原虫药物靶点,因为原生动物RNA三磷酸酶的机制和结构与哺乳动物和节肢动物宿主中发现的RNA三磷酸酶完全不同。在这里,我们报告了在活性位点隧道中与二价阳离子和无机三聚磷酸盐复合的 TcCet1 催化活性形式的几种晶体结构,分辨率为 2.20–2.51 Å。这些结构揭示了隧道的整体结构、建筑结构、TcCet1 中金属结合位点的排列与其他 TTM 蛋白中的相似。根据在蛋白质带正电荷的表面上共结晶的三个硫酸根离子的位置以及突变分析获得的结果,我们鉴定了 TcCet1 中的 RNA 结合位点。我们得出结论,三磷酸 RNA 底物的 5' 端定向进入活性位点隧道。这里报道的结构信息为设计抑制剂提供了宝贵的见解,这些抑制剂可以特异性阻止三磷酸RNA底物进入克氏锥虫和相关病原体的TTM型RNA三磷酸酶。根据在蛋白质带正电荷的表面上共结晶的三个硫酸根离子的位置以及突变分析获得的结果,我们鉴定了 TcCet1 中的 RNA 结合位点。我们得出结论,三磷酸 RNA 底物的 5' 端定向进入活性位点隧道。这里报道的结构信息为设计抑制剂提供了宝贵的见解,这些抑制剂可以特异性阻止三磷酸RNA底物进入克氏锥虫和相关病原体的TTM型RNA三磷酸酶。根据在蛋白质带正电荷的表面上共结晶的三个硫酸根离子的位置以及突变分析获得的结果,我们鉴定了 TcCet1 中的 RNA 结合位点。我们得出结论,三磷酸 RNA 底物的 5' 端定向进入活性位点隧道。这里报道的结构信息为设计抑制剂提供了宝贵的见解,这些抑制剂可以特异性阻止三磷酸RNA底物进入克氏锥虫和相关病原体的TTM型RNA三磷酸酶。
更新日期:2020-07-03
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