相关实验视频
Updated: Sep 30, 2026

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RhoC GTPase Activation Assay
Published on: August 22, 2010
在RhoA的1.65A结构及其GTPase激活蛋白与过渡状态模拟复合体中的结构
K Rittinger1, P A Walker, J F Eccleston
1National Institute for Medical Research, London, UK.
Nature
|October 24, 1997
概括
像RhoA这样的小G蛋白通过在GTP和GDP状态之间切换来调节细胞功能. 罗GAP加速了GTP水解,其Arg85残留稳定了GTP酶活性的过渡状态.
科学领域:
- 分子生物学分子生物学
- 结构生物学 结构生物学
- 生物化学 生物化学
背景情况:
- 罗家族的小G蛋白 (Rho,Rac,Cdc42Hs) 是细胞过程的关键调节者.
- 这些蛋白质起着分子开关的作用,在活跃的GTP结合和不活跃的GDP结合状态之间循环.
- 通过Rho蛋白进行GTP水解是缓慢的,但由GTP酶激活蛋白 (GAPs) 显著加速,例如rhoGAP.
研究的目的:
- 为了确定RhoA和rhoGAP复合物的晶体结构.
- 阐明rhoGAP刺激Rho蛋白中的GTP水解的机制.
- 调查GTPase活动期间过渡状态稳定性的结构基础.
主要方法:
- 通过X射线晶体学,获得了RhoA和rhoGAP复合物的结构.
- 该复合体以1.65 Å分辨率的过渡状态模拟GDP.AlF4-结晶.
- 与基态复合体 (Cdc42Hs.GMPPNP/rhoGAP) 进行了比较.
主要成果:
- 确定了RhoA-rhoGAP-GDP.AlF4复合物的晶体结构.
- 与基态复合体相比,在过渡状态复合体中观察到Rho和rhoGAP之间的20度旋转.
- 一个关键的发现是rhoGAP残留物Arg85的贡献直接进入过渡状态中的RhoA活性位点.
结论:
- 罗GAP残留物Arg85可能稳定了GTPase反应的过渡状态.
- 似乎RhoGAP稳定了参与GTP水解信号的RhoA的关键区域.
- 这种结构洞察力澄清了rhoGAP在调节Rho介导信号通路中的关键作用.
相关概念视频
GTPases and their Regulation
Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒ small G-proteins consisting of a single domain and large multi-domain G-proteins.
Large G-proteins, also known...
Large G-proteins, also known...
Rab Proteins
Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
GTPases and their Regulation
Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒ small G-proteins consisting of a single domain and large multi-domain G-proteins.
Large G-proteins, also known...
Large G-proteins, also known...
Rab Cascades
Rab GTPases act in a regulated cascade during membrane fusion, helping the lipid bilayers mix. The Rab family of proteins are active when bound to GTP, and inactive when bound to GDP. Hence, they act as guanine nucleotide-dependent molecular switches. Rab-GTP recognizes and binds to long or short-range tethering proteins to capture the target vesicle. These tethers coordinate with SNAREs on the vesicle and the target membrane to assemble the trans SNARE complex that locks the mixing bilayers.
Small GTPases - Ras and Rho
Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
Three regulatory proteins control their activity:
Activation and Inactivation of G Proteins
Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The Gα subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the Gꞵ and Gγ subunits are always bound together with high affinity and are together...

