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

含有激酶结构域的Rho-激酶和盘绕螺旋结构域的一部分由杆状病毒系统作为粘膜蛋白和谷胱甘肽-S-转移酶表示。该蛋白质通过几个色谱柱的步骤纯化，并最终以单个带，用Coomassie Brillial Blue染色，在SDS聚丙烯酰胺凝胶电泳中染色。我们收集了X射线小角度散射数据以检查蛋白质。数据显示蛋白质溶液是单分散的，蛋白质在溶液中形成四聚体。所有结晶实验均使用24孔组织培养板（Sumitomo Bakelite Co.）使用悬挂式蒸气 - 扩散法进行。我们广泛搜索了各种结晶条件，但直到现在才能获得晶体。 ERM（Ezrin/radixin/Moesin）蛋白识别膜相关细胞骨架的形成中粘合分子的细胞质结构域。我们报告使用ICAM-2 Cytop完成的radixin FERM结构域的晶体结构……更多的链肽。基于晶体结构的突变分析揭示了确定的识别元素，并提供了对粘合分子与ERM蛋白之间物理联系的首次见解。2E型（NF2）是与中枢神经系统相关的主要遗传疾病。 NF2基因产物Merlin是一种肿瘤抑制剂，其突变或灭活会导致这种疾病。我们在这里报告了含有22个残基螺旋段的Merlin Ferm结构域的晶体结构。该结构表明，Merlin FERM结构域由三个子域组成，这些子域显示了静电表面电位的显着特征，尽管总体表面电位与ERM蛋白相似，表明其静电膜关联。该结构还表明，与NF2相关的致病突变引起的灭活机制。此外，我们得到了phogdi的晶体符合ERM蛋白。结构确定正在进行中。较少的

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。