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

含有激酶结构域和部分卷曲螺旋结构域的 Rho 激酶通过杆状病毒系统表达为与谷胱甘肽-S-转移酶的融合蛋白。该蛋白通过多个柱层析步骤纯化，最终获得单条带，并用考马斯染色。亮蓝色，在 SDS 聚丙烯酰胺凝胶电泳中我们收集了 X 射线小角散射数据来检查蛋白质，数据显示蛋白质溶液是单分散的。所有结晶实验均采用24孔组织培养板（住友电木有限公司）通过悬滴蒸气扩散法进行，主要探索了各种结晶条件，但至今未能获得晶体。 ERM（ezrin/radixin/moesin）蛋白在膜相关细胞骨架的形成过程中识别粘附分子的细胞质结构域我们报告了 radixin FERM 结构域的晶体结构。使用 ICAM-2 细胞质肽完成基于晶体结构的突变分析揭示了识别的决定因素，并首次深入了解粘附分子和 ERM 蛋白之间的物理联系。2 型神经纤维瘤病 (NF2) 是一种主要的疾病。与中枢神经系统相关的遗传性疾病。 NF2 基因产物 merlin 是一种肿瘤抑制因子，其突变或失活会导致这种疾病。我们在此报告包含 merlin FERM 结构域的晶体结构。该结构揭示了 merlin FERM 结构域由三个子结构域组成，显示出静电表面电位的显着特征，尽管整体表面电位与 ERM 蛋白相似，表明其静电膜关联。与NF2相关的致病性突变引起的失活机制。此外，我们得到的符合ERM蛋白的PhoGDI晶体的结构测定正在进行中。

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 细胞质尾部结合的根蛋白 FERM 结构域的晶体学特征”Acta Crystallogr.，D57。

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。