Structural basis for auto-inhibition mechanism of Rho-kinase

Rho激酶自抑制机制的结构基础

• 批准号: 13680742
• 负责人: SHIMIZU Toshiyuki
• 金额: $ 2.3万
• 依托单位: Yokohama City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680742/
• 关键词: Rho-kinase; Rho-signalling; auto-inhibition; X-ray analysis; tertiary structure; ERM protein; Merlin; FERM domain; Rho-キナーゼ; コイルドコイル; DHドメイン; Rho

Rho-kinase containing Kinase domain and part of coiled-coil domain is expressed by baculovirus systems as a fusion protein with glutathione-S-transferase. The protein is purified by several column chromatography steps and was finally obtained as a single band, stained with Coomassie brilliant blue, in SDS polyacrylamide gel electrophoresis. We collected X-ray small angle scattering data to check the protein. The data show the protein solution is mono-disperse and protein forms tetramer in solution. All the crystallization experiments were carried out with the hanging-drop vapor-diffusion method using 24-well tissue-culture plates (sumitomo Bakelite Co.). We searched various crystallization conditions extensively, but failed to get crystals until now. ERM (ezrin/radixin/moesin) proteins recognize the cytoplasmic domain of adhesion molecules in the formation of the membrane-associated cytoskeleton. We report the crystal structure of the radixin FERM domain completed with the ICAM-2 cytop … More lasmic peptide. Mutations analyses based on the crystal structure reveal the determinant elements of recognition and provide the first insight into the physical link between adhesion molecules and ERM proteins.Neurofibromatosis type 2 (NF2) is a dominantly inherited disease associated with the central nervous system. The NF2 gene product merlin is a tumor suppressor and its mutation or inactivation causes this disease.We report here the crystal structure of the merlin FERM domain containing a 22-residue a helical segment. The structure reveals that the merlin FERM domain consists of three subdomains displaying notable features of the electrostatic surface potentials, although the overall surface potentials similar to those of ERM proteins indicate its electrostatic membrane association. The structure also suggests the inactivation mechanisms caused by the pathogenic mutations associated with NF2.Moreover, we get the crystals of PhoGDI complied with ERM protein. Structure determination is in progress. Less

含有激动区和部分卷曲结构域的Rho-Kinase在杆状病毒系统中以谷胱甘肽-S-转移酶融合蛋白的形式表达。该蛋白经几个柱层析步骤纯化后，经考马斯亮蓝染色，经SDS-聚丙烯酰胺凝胶电泳法得到单一条带。我们收集了X射线小角散射数据来检查蛋白质。结果表明，蛋白质溶液呈单分散状态，蛋白质在溶液中形成四聚体。采用24孔组织培养板(住友胶木株式会社)，采用悬滴气相扩散法进行结晶实验。我们对各种结晶条件进行了广泛的搜索，但到目前为止都没有得到结晶。ERM(ezrin/Radioxin/moesin)蛋白识别膜相关细胞骨架形成过程中黏附分子的细胞质结构域。我们报道了用ICAM-2 Cytop…完成的Radioxferm结构域的晶体结构更多的拉西多肽。基于晶体结构的突变分析揭示了识别的决定因素，并首次揭示了黏附分子和ERM蛋白之间的物理联系。神经纤维瘤病2型(NF2)是一种与中枢神经系统相关的主要遗传性疾病。NF2基因产物Merlin是一种肿瘤抑制基因，它的突变或失活会导致这种疾病。我们在这里报道了包含22个残基a螺旋片段的Merlin费米域的晶体结构。结构显示Merlin Ferm结构域由三个亚域组成，显示出显著的静电表面电位特征，尽管总体表面电位与ERM蛋白相似，表明其静电膜结合。该结构还提示了与NF2相关的致病突变导致的失活机制。此外，我们还得到了符合ERM蛋白的PhoGDI晶体。结构确定正在进行中。较少

项目成果

Shimizu, T.: "Auto-inhibition mechanism of proteins in signal transduction"PEN. 46. 1950-1955 (2001)

Shimizu, T.：“信号转导中蛋白质的自动抑制机制”PEN。

Hamada, K. et al.: "Crystallization and preliminary crystallographic studies of RhoGDI in complex with the radixin FERM domain"Acta Crystallogr. D57. 889-890 (2001)

Hamada, K. 等人：“RhoGDI 与根素 FERM 结构域复合物的结晶和初步晶体学研究”Acta Crystallogr。

清水: "転写因子の構造生物学"細胞工学. 20. 1359-1363 (2001)

清水：“转录因子的结构生物学”细胞工程20。1359-1363（2001）。

Hamada, K. et al.: "Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of the adhesion protein ICAM-2"Acta Crystallogr. D57. 890-891 (2001)

Hamada, K. 等人：“与粘附蛋白 ICAM-2 细胞质尾部结合的 radixin FERM 结构域的晶体学特征”Acta Crystallogr。

Hamada, K. et al: "Crystallization and preliminary crystallographic studies of RhoGDI in complex with the radixin FERM domain"Acta Crystallogr. D57. 889-890 (2001)

Hamada, K. 等人：“RhoGDI 与根素 FERM 结构域复合物的结晶和初步晶体学研究”Acta Crystallogr。