Histidine triad-like motif of the rotavirus NSP2 octamer mediates both RTPase and NTPase activities
Histidine triad-like motif of the rotavirus NSP2 octamer mediates both RTPase and NTPase activities
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DOI：
10.1016/j.jmb.2006.07.050
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发表时间：
2006-09-22
影响因子：
5.6
通讯作者：
Patton, John T.
中科院分区：
文献类型：
--
作者：
Carpio, Rodrigo Vasquez-Del;Gonzalez-Nilo, Fernando D.;Patton, John T.
轮状病毒NSP 2是一种丰富的非结构RNA结合蛋白，对于形成支持双链RNA基因组复制的病毒工厂至关重要。NSP 2在感染细胞内以稳定的甜甜圈形八聚体存在，代表两个四聚体的尾对尾相互作用。每个八聚体表面的对角延伸是四个高度碱性的凹槽，作为单链RNA的结合位点。在每个单体的N和C末端结构域之间是一个深的正电性裂缝，该裂缝含有水解任何NTP的γ-β磷酸酐键的催化位点。催化位点与核苷酸结合蛋白的组氨酸三联体（HIT）家族的催化位点具有相似性。由于凹槽和裂缝的紧密接近，我们研究了凹槽的RNA结合活性促进RNA的5 '-三磷酸部分插入裂缝中以及随后其γ-β磷酸酐键水解的可能性。我们的研究结果表明，NSP 2水解的γ P从RNA和NTPs通过Mg 2+依赖的活动，进行类似的反应速度，需要催化的His 225残基，并产生磷酸化的中间体。竞争分析表明，虽然两种底物都进入活性位点，但RNA是优选的底物，因为其对八聚体具有更高的亲和力。NSP 2的RNA三磷酸酶（RTP 3）活性可以解释双链RNA基因组区段的（-）链上5 '-末端γ P的缺失。这是第一次报告的HIT样蛋白具有多功能的催化位点，能够同时容纳NTPs和RNA在γ P水解。爱思唯尔有限公司出版
Rotavirus NSP2 is an abundant non-structural RNA-binding protein essential for forming the viral factories that support replication of the double-stranded RNA genome. NSP2 exists as stable doughnut-shaped octamers within the infected cell, representing the tail-to-tail interaction of two tetramers. Extending diagonally across the surface of each octamer are four highly basic grooves that function as binding sites for single-stranded RNA. Between the N and C-terminal domains of each monomer is a deep electropositive cleft containing a catalytic site that hydrolyzes the gamma-beta phosphoanhydride bond of any NTP. The catalytic site has similarity to those of the histidine triad (HIT) family of nucleotide-binding proteins. Due to the close proximity of the grooves and clefts, we investigated the possibility that the RNA-binding activity of the groove promoted the insertion of the 5'-triphosphate moiety of the RNA into the cleft, and the subsequent hydrolysis of its gamma-beta phosphoanhydride bond. Our results show that NSP2 hydrolyzes the gamma P from RNAs and NTPs through Mg2+ -dependent activities that proceed with similar reaction velocities, that require the catalytic His225 residue, and that produce a phosphorylated intermediate. Competition assays indicate that although both substrates enter the active site, RNA is the preferred substrate due to its higher affinity for the octamer. The RNA triphosphatase (RTPase) activity of NSP2 may account for the absence of the 5'-terminal gamma P on the (-) strands of the double-stranded RNA genome segments. This is the first report of a HIT-like protein with a multifunctional catalytic site, capable of accommodating both NTPs and RNAs during gamma P hydrolysis. Published by Elsevier Ltd.