Partial proteolysis of Simian virus 40 T antigen reveals intramolecular contacts between domains and conformation changes upon hexamer assembly
Partial proteolysis of Simian virus 40 T antigen reveals intramolecular contacts between domains and conformation changes upon hexamer assembly
复制标题
DOI：
10.1074/jbc.m406159200
复制
发表时间：
2004-09-10
影响因子：
4.8
通讯作者：
Fanning, E
中科院分区：
文献类型：
--
作者：
Weisshart, K;Friedl, S;Fanning, E
猴病毒40大肿瘤抗原（Tag）是一种多功能病毒蛋白，其特异性结合SV40起源DNA，充当复制DNA解旋酶，并协调病毒复制体的组装和操作。与Mg-ATP相关的标签形成六聚体，并且在SV40来源DNA存在下形成双六聚体。有限的胰蛋白酶消化的单体标签显示三个主要的稳定结构域。N-末端结构域跨越氨基酸1 - 130，中心结构域包含氨基酸131 - 476，并且C-末端结构域从氨基酸513延伸至氨基酸698。单体标签的消化产物的免疫共沉淀表明，N-末端结构域与位于相同标签分子的中心区域的序列稳定地关联。六聚体的形成保护了中央和C-末端结构域之间的暴露区域中的胰蛋白酶切割位点。在六聚化后，该暴露区域也变得不太容易接近其表位定位在该区域中的单克隆抗体。可溶性六聚体和DNA结合的双六聚体的胰蛋白酶消化产物是不可区分的。提出了双六聚体中Tag结构域之间分子内和分子间相互作用的低分辨率模型。
Simian virus 40 large tumor antigen ( Tag) is a multifunctional viral protein that binds specifically to SV40 origin DNA, serves as the replicative DNA helicase, and orchestrates the assembly and operation of the viral replisome. Tag associated with Mg-ATP forms hexamers and, in the presence of SV40 origin DNA, double hexamers. Limited tryptic digestion of monomeric Tag revealed three major stable structural domains. The N-terminal domain spans amino acids 1 - 130, the central domain comprises amino acids 131 - 476, and the C-terminal domain extends from amino acid 513 to amino acid 698. Co-immunoprecipitation of digestion products of monomeric Tag suggests that the N-terminal domain associates stably with sequences located in the central region of the same Tag molecule. Hexamer formation protected the tryptic cleavage sites in the exposed region between the central and C-terminal domains. Upon hexamerization, this exposed region also became less accessible to a monoclonal antibody whose epitope maps in that region. The tryptic digestion products of the soluble hexamer and the DNA-bound double hexamer were indistinguishable. A low-resolution model of the intramolecular and intermolecular interactions among Tag domains in the double hexamer is proposed.