Affinity regulation mechanism of microtubule-binding domain of cytoplasmic dynein

细胞质动力蛋白微管结合域的亲和力调节机制

• 批准号: 24770090
• 负责人: NISHIDA Noritaka
• 金额: $ 3万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2012
• 资助国家: 日本
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-24770090/
• 关键词: 核磁気共鳴法; クライオ電顕; モーター蛋白質; 電子顕微鏡; 分子モーター

Cytoplasmic dynein is a motor protein that walks on microtubules (MTs). The microtubule-binding domain (MTBD) is located at the tip of the anti-parallel coiled-coil, which is protruded from the ATPase domain of dynein heavy chain. It has been considered that the change in the association mode of the coiled-coil alters the MT affinity of the MTBD. However, the structural mechanism underlying the affinity regulation of the MTBD remains elusive.Here, we designed two MTBD constructs, termed MTBD-High and MTBD-Low, which are stabilized either in the high or low affinity state for MTs, respectively, by introducing a disulfide bond between the coiled-coil helix. We performed NMR analyses of MTBD-High and MTBD-Low, in order to elucidate differences in the structure and the MT-binding mode in the high and low affinity states. Based on NMR data in combination with the cryoEM analysis of the MTBD-High/MTs complex, we modeled the complex structure of the MTBD and MTs in the strong binding state.

细胞质动力蛋白是一种在微管上行走的运动蛋白。微管结合结构域（MTBD）位于反平行卷曲螺旋的末端，从动力蛋白重链的ATP酶结构域突出。已经认为卷曲螺旋的缔合模式的改变改变了MTBD的MT亲和力。然而，MTBD的亲和力调节的结构机制仍然难以捉摸，在这里，我们设计了两个MTBD构建体，称为MTBD-High和MTBD-Low，它们通过在卷曲螺旋螺旋之间引入二硫键分别稳定在对MT的高或低亲和力状态。我们对MTBD-高和MTBD-低进行了NMR分析，以阐明高和低亲和力状态下结构和MT结合模式的差异。基于核磁共振数据并结合MTBD-高/MT复合物的cryoEM分析，我们对MTBD和MT在强结合状态下的复合物结构进行了建模。

项目成果

NMR observation of protein-protein interactions in living mammalian cells using a gel encapsulated bioreactor system

使用凝胶封装生物反应器系统对活哺乳动物细胞中蛋白质-蛋白质相互作用进行核磁共振观察

• 发表时间: 2013
• 作者: 西田紀貴;嶋田一夫
• 通讯作者: 嶋田一夫

An NMR method to observe protein・protein interactions in living mammalian cells using the gel encapsulated bioreactor system

使用凝胶封装生物反应器系统观察活哺乳动物细胞中蛋白质与蛋白质相互作用的 NMR 方法

• 发表时间: 2012
• 作者: Satoshi Kubo;Noritaka Nishida;Yuko Udagawa;Osamu Takarada;Shinji Ogino;Ichio Shimada;久保 智史
• 通讯作者: 久保 智史

A gel-encapsulated bioreactor system for NMR studies of protein-protein interactions in living mammalian cells. ゲル包埋型バイオリアクターを用いた生細胞内蛋白質間相互作用のNMR観測

用于活哺乳动物细胞中蛋白质-蛋白质相互作用的核磁共振研究的凝胶封装生物反应器系统。

• 发表时间: 2013
• 作者: 西田紀貴;嶋田一夫
• 通讯作者: 嶋田一夫

Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins.

• DOI: 10.1016/j.jbiosc.2013.02.012
• 发表时间: 2013-08
• 期刊: Journal of bioscience and bioengineering
• 影响因子: 2.8
• 作者: N. Nishida;M. Yagi-Utsumi;F. Motojima;Masasuke Yoshida;I. Shimada;Koichi Kato

Affinity regulation mechanism of microtubule-binding domain of cytoplasmic dynein

细胞质动力蛋白微管结合域的亲和力调节机制

• 发表时间: 2013
• 作者: 西田紀貴;宝田理;吉川雅英;嶋田一夫
• 通讯作者: 嶋田一夫