Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications
Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications
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DOI：
10.1074/jbc.m109.090936
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发表时间：
2010-06-11
影响因子：
4.8
通讯作者：
Vasudevan, Subhash G.
中科院分区：
文献类型：
--
作者：
Luo, Dahai;Wei, Na;Vasudevan, Subhash G.
登革病毒（DENV）NS 3蛋白是病毒多蛋白加工和RNA复制所必需的。它含有一个N-末端丝氨酸蛋白酶区（残基1-168），通过一个11-氨基酸接头（169-179）与RNA解旋酶（残基180-618）连接。来自DENV 4的可溶性NS 3蛋白在3.15埃处的结构共价连接到NS 2B辅因子区的18个残基（NS 2B（18）NS 3），揭示了具有邻接解旋酶的亚结构域I和II的蛋白酶结构域的伸长分子（Luo，D.，徐，T.，亨克角，格鲁伯，G.，Vasudevan，S. G.，和莱斯卡尔，J.（2008）J. Virol. 82，173-183）。出乎意料的是，使用类似的晶体生长条件，我们观察到一种替代构象，其中蛋白酶结构域相对于解旋酶结构域旋转了类似于161度。我们报告了这个新的晶体结构绑定到ADP-Mn 2+精制到2.2埃的分辨率。通过在Glu（173）和Pro 174之间插入Gly残基或用Gly残基取代Pro 174来探测由接头区赋予的结构域间柔性的生物学意义。两种突变均导致ATP酶和解旋酶活性显著降低。我们接下来通过在DENV 2复制子系统中引入Pro（176）至Gly突变来增加接头的灵活性。观察到荧光素酶报告信号减少70%，病毒RNA合成水平也类似减少。我们的研究结果表明，接头区域已发展到一个最佳的长度，赋予灵活性的NS 3蛋白，这是所需的多蛋白加工和RNA复制。
The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1-168) joined to an RNA helicase (residues 180-618) by an 11-amino acid linker (169-179). The structure at 3.15 angstrom of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B(18)NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Gruber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173-183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by similar to 161 degrees with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn2+ refined to a resolution of 2.2 angstrom. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu(173) and Pro174 or replacing Pro174 with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro(176) to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.