School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
PLoS One. 2010 Sep 17;5(9):e12836. doi: 10.1371/journal.pone.0012836.
The N-terminal domain of the flavivirus NS5 protein functions as a methyltransferase (MTase). It sequentially methylates the N7 and 2'-O positions of the viral RNA cap structure (GpppA→(7me)GpppA→(7me)GpppA(2'-O-me)). The same NS5 domain could also have a guanylyltransferase activity (GTP+ppA-RNA→GpppA). The mechanism by which this protein domain catalyzes these three distinct functions is currently unknown. Here we report the crystallographic structure of DENV-3 MTase in complex with a 5'-capped RNA octamer (G(ppp)AGAACCUG) at a resolution of 2.9 A. Two RNA octamers arranged as kissing loops are encircled by four MTase monomers around a 2-fold non-crystallography symmetry axis. Only two of the four monomers make direct contact with the 5' end of RNA. The RNA structure is stabilised by the formation of several intra and intermolecular base stacking and non-canonical base pairs. The structure may represent the product of guanylylation of the viral genome prior to the subsequent methylation events that require repositioning of the RNA substrate to reach to the methyl-donor sites. The crystal structure provides a structural explanation for the observed trans-complementation of MTases with different methylation defects.
黄病毒 NS5 蛋白的 N 端结构域具有甲基转移酶（MTase）的功能。它依次对病毒 RNA 帽结构的 N7 和 2'-O 位置进行甲基化（GpppA→(7me)GpppA→(7me)GpppA(2'-O-me)）。同一 NS5 结构域也具有鸟苷转移酶活性（GTP+ppA-RNA→GpppA）。目前尚不清楚该蛋白结构域催化这三种不同功能的机制。本文报道了 DENV-3 MTase 与 5'-加帽 RNA 八聚体（G(ppp)AGAACCUG）复合物的晶体结构，分辨率为 2.9 A。两个以亲吻环形式排列的 RNA 八聚体被四个 MTase 单体围绕在 2 倍非晶学对称轴周围。只有四个单体中的两个与 RNA 的 5' 端直接接触。RNA 结构通过形成几个分子内和分子间碱基堆积和非规范碱基对得到稳定。该结构可能代表病毒基因组鸟苷化的产物，随后是需要重新定位 RNA 底物以到达甲基供体位点的后续甲基化事件。晶体结构为观察到的具有不同甲基化缺陷的 MTases 的转互补提供了结构解释。
