Structure-based design of a disulfide-linked oligomeric form of the simian virus 40 (SV40) large T antigen DNA-binding domain.
Structure-based design of a disulfide-linked oligomeric form of the simian virus 40 (SV40) large T antigen DNA-binding domain.
复制标题
基于结构的猿猴病毒 40 (SV40) 大 T 抗原 DNA 结合结构域的二硫键连接寡聚形式的设计。
DOI：
10.1107/s0907444911014302
复制
发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Bullock,PeterA
中科院分区：
文献类型：
--
作者：
Meinke,Gretchen;Phelan,Paul;Fradet-Turcotte,Amélie;Archambault,Jacques;Bullock,PeterA
来自猿猴病毒 40 的模块化多功能蛋白大 T 抗原 (T-ag) 协调了病毒双链 DNA 基因组复制所需的许多事件。该蛋白质在位于复制起点的特定 DNA 序列上组装成单六聚体和双六聚体。当大 T 抗原 (T-ag ODB) 的起点结合域与病毒复制起点中心区域（位点 II）的 GAGGC 序列结合时，这个复杂的过程就开始了。虽然纯化的 T-ag OBD 的许多功能可以单独研究，但它在溶液中主要是单体，不能组装成六聚体。为了克服这一限制，研究了在起始结合域中设计分子间二硫键的可能性，该二硫键可以在溶液中寡聚。野生型 T-ag OBD 的最新晶体结构表明，该结构域在晶体中形成左手螺旋，每圈有 6 个亚基。因此，我们分析了该结构的蛋白质界面，并鉴定了两个残基，如果更改为半胱氨酸，则可能支持分子间二硫键。 SDS-PAGE 分析表明，突变型 T-ag OBD 以氧化还原依赖性方式形成更高的寡聚产物。此外，还报道了工程化二硫键连接的 T-ag OBD 的 1.7 Å 分辨率晶体结构，这证实了寡聚化以预期的方式发生。
The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of the viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication. While many of the functions of purified T-ag OBD can be studied in isolation, it is primarily monomeric in solution and cannot assemble into hexamers. To overcome this limitation, the possibility of engineering intermolecular disulfide bonds in the origin-binding domain which could oligomerize in solution was investigated. A recent crystal structure of the wild-type T-ag OBD showed that this domain forms a left-handed spiral in the crystal with six subunits per turn. Therefore, we analyzed the protein interface of this structure and identified two residues that could potentially support an intermolecular disulfide bond if changed to cysteines. SDS–PAGE analysis established that the mutant T-ag OBD formed higher oligomeric products in a redox-dependent manner. In addition, the 1.7 Å resolution crystal structure of the engineered disulfide-linked T-ag OBD is reported, which establishes that oligomerization took place in the expected manner.