兰州大学机构库 >生命科学学院
电镜单颗粒技术研究全长AMPK蛋白的构架及变构效应
Alternative TitleArchitecture and allostery of full length AMP-activated protein kinase by single particle electron microscopy
朱莉
Thesis Advisor武一
2011-05-28
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
KeywordAMP活化的蛋白激酶 负染 电镜单颗粒三维重构 寡聚
Abstract哺乳动物AMP活化的蛋白激酶(简称AMPK)是由α、β、γ三个亚基组成的异三聚体蛋白质复合物,可以灵敏地感知胞内AMP/ATP水平的变化,在维持真核细胞及有机体的代谢及能量的动态平衡中起着非常重要的作用。其中α为催化亚基,β为脚手架亚基,γ为调控亚基,含有3个腺苷结合位点,其中两个可以竞争性地结合ATP•Mg或AMP,第三个位点结合一分子不可交换的AMP。AMPK可由上游蛋白激酶将其α亚基activation loop上保守的Thr172位点磷酸化而激活,加上AMP与γ亚基结合产生的变构激活作用,可以使得激酶活性增加1000倍。本论文中我们首次报导了由电镜单颗粒三维重构技术解析出的鼠源全长AMPK同源三聚体在基态及不同腺苷结合状态的三维结构,讨论了AMPK形成三聚体的寡聚条件及介导其寡聚的因素,确定了全长AMPK的分子构架方式。观察到了腺苷与γ亚基结合后导致α亚基的激酶结构域被抑制或激活的这一过程中的一系列构象变化。最后,我们给出了AMPK结构及其调控的三态模型。本论文的发现不仅对当前的AMPK组装模型做出了较大修正,而且揭示了α亚基连接序列(LS)在感知γ亚基配体结合状态和传递/放大亚基间长程变构效应中的关键作用及可能机制,同时对进一步研究AMPK亚基内各个结构域或亚基间可能存在的相互作用提供了一定的思路。
Other AbstractMammalian AMP-activated protein kinase (AMPK) is a heterotrimeric protein complex, consisting of α catalytic subunit, β scaffolding subunit and γ regulatory subunit. It’s a master intracellular sensor for the changes in the ratio of AMP/ATP, and has a crucial role in maintaining the metabolism and cellular energy homeostasis of eukaryotic cells and organisms. The γ subunit contains three adenosine binding sites, two of which are exchangeable for ATP•Mg and AMP while the third site contains a tightly bound AMP that does not exchange. AMPK is activated (~1000 fold) by phosphorylation of a conserved threonine (Thr172 in rat) in the activation loop of the KD by upstream kinases, together with AMP binding to its γ subunits. In our work, we report the first 3D reconstruction of full length rat AMPK in basal and adenosine-bound states by single particle electron microscopy and have discussed the condition for AMPK monomers forming homotrimer, the possible electrostatic interaction mediating the oligomerization and the molecular architecture of AMPK. We are able to visualize the sequential conformational changes induced kinase activation or inhibition that transmits from the adenosine binding sites in the γ subunit to the kinase domain of the α subunit. Finally, we yield a three-state model for AMPK structure and regulation. Our findings not only make substantial revision to the current model of AMPK assembly, but also highlight a key role of the linker sequence of the α subunit in sensing the adenosine binding state of the γ subunit and the presumed mechanism in transmission and amplification of the long-term allostery of AMPK between the three subunits, meanwhile provide clues for further study of the inter- and intrasubunit cross-talks underlying the assembly and function of AMPK.
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221538
Collection生命科学学院
Recommended Citation
GB/T 7714
朱莉. 电镜单颗粒技术研究全长AMPK蛋白的构架及变构效应[D]. 兰州. 兰州大学,2011.
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