Vasquez-Del Carpio Rodrigo, Gonzalez-Nilo Fernando D, Riadi Gonzalo, Taraporewala Zenobia F, Patton John T
Laboratory of Infectious Diseases, NIAID, National Institutes of Health, Bethesda, MD 20892, USA.
J Mol Biol. 2006 Sep 22;362(3):539-54. doi: 10.1016/j.jmb.2006.07.050. Epub 2006 Jul 29.
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 gammaP from RNAs and NTPs through Mg(2+)-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 gammaP 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 gammaP hydrolysis.
轮状病毒NSP2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。NSP2在受感染细胞内以稳定的甜甜圈形状的八聚体形式存在，代表两个四聚体的尾对尾相互作用。在每个八聚体表面对角延伸的是四个高度碱性的凹槽，它们作为单链RNA的结合位点。在每个单体的N端和C端结构域之间是一个深的正电裂隙，其中含有一个催化位点，可水解任何NTP的γ-β磷酸酐键。该催化位点与组氨酸三联体（HIT）家族的核苷酸结合蛋白的催化位点相似。由于凹槽和裂隙靠得很近，我们研究了凹槽的RNA结合活性是否促进RNA的5'-三磷酸部分插入裂隙，并随后水解其γ-β磷酸酐键的可能性。我们的结果表明，NSP2通过Mg(2+)依赖性活性水解RNA和NTP中的γP，这些活性以相似的反应速度进行，需要催化性的His225残基，并产生磷酸化中间体。竞争试验表明，尽管两种底物都进入活性位点，但由于RNA对八聚体具有更高的亲和力，所以它是首选底物。NSP2的RNA三磷酸酶（RTPase）活性可能解释了双链RNA基因组片段（-）链上5'-末端γP的缺失。这是关于一种具有多功能催化位点的类HIT蛋白的首次报道，该蛋白在γP水解过程中能够容纳NTP和RNA。
