Yamamoto Takahiko, Matsuda Tomoki, Inoue Tsuyoshi, Matsumura Hiroyoshi, Morikawa Masaaki, Kanaya Shigenori, Kai Yasushi
Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
Protein Sci. 2006 Jan;15(1):152-61. doi: 10.1110/ps.051788906. Epub 2005 Dec 1.
TATA-binding protein (TBP)-interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1 (Tk-TIP26) is a possible transcription regulatory protein in Thermococcales. Here, we report the crystal structure of Tk-TIP26 determined at 2.3 A resolution with multiple-wavelength anomalous dispersion (MAD) method. The overall structure of Tk-TIP26 consists of two domains. The N-terminal domain forms an alpha/beta structure, in which three alpha-helices enclose the central beta-sheet. The topology of this domain is similar to that of holliday junction resolvase Hjc from Pyrococcus furiosus. The C-terminal domain comprises three alpha-helices, six beta-strands, and two 3(10)-helices. In the dimer structure of Tk-TIP26, two molecules are related with the crystallographic twofold axis, and these molecules rigidly interact with each other via hydrogen bonds. The complex of Tk-TIP26/Tk-TBP is isolated and analyzed by SDS-PAGE and gel filtration column chromatography, resulting in a stoichiometric ratio of the interaction between Tk-TIP26 and Tk-TBP of 4:2, i.e., two dimer molecules of Tk-TIP26 formed a complex with one dimeric TBP. The electrostatic surfaces of Tk-TIP26 and TBP from Pyrocuccus woesei (PwTBP) allowed us to build a model of the Tk-TIP26/TBP complex, and to propose the inhibition mechanism where two dimer molecules of Tk-TIP26 bind to a dimeric TBP, preventing its binding to TATA-DNA.
来自嗜热古菌柯达嗜热栖热菌菌株KOD1的TATA结合蛋白（TBP）相互作用蛋白（Tk-TIP26）可能是嗜热栖热菌目中的一种转录调节蛋白。在此，我们报告了采用多波长反常色散（MAD）方法以2.3埃分辨率测定的Tk-TIP26晶体结构。Tk-TIP26的整体结构由两个结构域组成。N端结构域形成α/β结构，其中三个α螺旋围绕着中央β折叠。该结构域的拓扑结构与来自激烈火球菌的霍利迪连接解离酶Hjc相似。C端结构域由三个α螺旋、六条β链和两个3（10）螺旋组成。在Tk-TIP26的二聚体结构中，两个分子通过晶体学二重轴相关，并且这些分子通过氢键彼此刚性相互作用。通过SDS-PAGE和凝胶过滤柱色谱法分离并分析Tk-TIP26/Tk-TBP复合物，结果表明Tk-TIP26与Tk-TBP之间相互作用的化学计量比为4:2，即两个Tk-TIP26二聚体分子与一个二聚体TBP形成复合物。Tk-TIP26和来自沃氏火球菌的TBP（PwTBP）的静电表面使我们能够构建Tk-TIP26/TBP复合物模型，并提出抑制机制，即两个Tk-TIP26二聚体分子与一个二聚体TBP结合，阻止其与TATA-DNA结合。
