【作者】 朱莉；

【导师】 武一；

【作者基本信息】 兰州大学 ， 生物物理， 2011， 博士

【摘要】 哺乳动物AMP活化的蛋白激酶(简称AMPK)是由α(两种亚型)、β(两种亚型)、γ(三种亚型)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,主要包括N端的丝/苏氨酸激酶结构域(KD)、推测可能具有自抑制功能的AID结构域以及C端与β亚基结合的结构域。β为脚手架亚基,其N端的肉豆蔻酰基团可以介导AMPK与膜相互作用,中间为糖原结合结构域(GBD),而C端通过与α及γ亚基的结合,介导了整个AMPK的组装。γ为调控亚基,由四个串联的CBS结构域组成两对Bateman结构域,圆盘状的结构中央有一个小的可溶性通道,并含有3个腺苷结合位点,其中两个可以竞争性地结合ATP·Mg2+或AMP,第三个位点牢固地结合着一分子不可交换的AMP。三亚基结构的完整性是AMPK发挥正常激酶活性的前提。AMPK可由上游蛋白激酶LKB1-MO25α-STRADα或CaMKKβ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。尽管已经由X射线晶体衍射技术解析出了AMPK蛋白大部分序列片段的晶体结构,但由于缺失完整复合物的结构信息,因此还有一些问题目前仍未得到解决。首先,全长AMPK三亚基的构架是未知的；其次,晶体结构中腺苷与γ亚基结合产生的微小的构象变化是如何传递至α亚基并调控其激酶活性的也仍不清楚；还有,有关AMPK同源寡聚体的存在及功能已有文献报导,但关于寡聚形式以及介导其寡聚的因素仍值得进一步探讨。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蛋白(亚型组成为α1β1γ1)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。通过构建AMPK的激酶结构域(KD)和糖原结合结构域(GBD)的删截突变体,辅助以晶体结构的拟合计算,我们确定了全长AMPK的分子构架方式。经过对AMPK+ATP模型和AMPK+AMP模型的比较,我们观察到了腺苷与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。我们对AMPK+ATP模型与AMPK基态模型比较后得出的结论是,AMPK在第1、2腺苷结合位点未有结合时,其构象趋于处在变构的激发状态。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。更多还原

【Abstract】 Mammalian AMP-activated protein kinase (AMPK) is a heterotrimeric protein complex, consisting ofα(two isoforms),β(two isoforms) andγ(three isoforms) subunits. 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 a catalytic subunit contains an N-terminal Ser/Thr kinase domain (KD), a putative autoinhibitory domain (AID) and a C-terminal region required forβsubunit binding. Theβscaffolding subunit mediates AMPK assembly by binding with bothαandγsubunits through its C-terminal sequence and also contains an N-terminal myristoyl group which is responsible for targeting AMPK to the membrane and an internal glycogen-binding domain. Theγregulatory subunit, composed of four tandem cystathionineβ-synthase (CBS) domains which making up two pairs of Bateman domains, is shaped like a flattened disk with a small solvent-accessible channel around the center, and contains three adenosine binding sites, two of which are exchangeable for ATP·Mg2+ and AMP while the third site contains a tightly bound AMP that does not exchange. The integrity of all these three subunits is the precondition of AMPK to get optimal activity. AMPK is activated (～1000 fold) by phosphorylation of a conserved threonine (Thr172 in rat) in the activation loop of the KD by upstream kinases, including LKB1-MO25α-STRADαor CaMKKβ, and together with AMP binding to itsγsubunits.Although published X-ray crystal structures have covered most of AMPK’s sequence, several fundamental issues on AMPK’s function still remain elusive due to lacking structural information of the intact complex. First, the molecular architecture of the three multidomain subunits in AMPK is unknown. Second, it is unclear how the subtle structural changes seen in the crystal structure of theγsubunits upon adenosine binding can be ultimately translated into the regulation of the kinase activity in theαsubunit. Finally, whereas the existence and functional relevance of AMPK homo-oligomer is emerging, it is necessary to further dissect the structure and determinants of AMPK oligomerization. In our work, we report the first 3D reconstruction of full length rat AMPK (α1β1γ1) in basal and adenosine-bound states by single particle electron microscopy and have discussed the condition for AMPK monomers forming homotrimer and the possible electrostatic interaction mediating the oligomerization.With the aid of observation of the AMPK truncation mutants, and docking of the X-ray crystal structures of AMPK subunits or fragments, we are able to establish the molecular architecture of AMPK. By comparing the structures of AMPK in ATP-and AMP-bound states, we are able to visualize the sequential conformational changes induced kinase activation or inhibition that transmits from the adenosine binding sites in theγsubunit to the kinase domain of theαsubunit. And by comparing AMPK in ATP-bound state with its basal state, we conclude that AMPK in basal state may actually be in a priming configuration. Finally, we yield a three-state model for AMPK structure and regulation.Our findings reported here not only make substantial revision to the current model of AMPK assembly, but also highlight a key role of the linker sequence of theαsubunit in sensing the adenosine binding state of theγsubunit 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.更多还原