【作者】 赵梦溪；

【导师】 周丛照； 陈宇星；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 蓝细菌,又称蓝绿藻,是一种可以进行光合作用的原核生物。一些蓝细菌,如丝状蓝细菌鱼腥蓝细菌,还可以进行固氮作用。鱼腥蓝细菌生长在淡水环境中,在营养丰盛的条件下,藻丝中细胞以营养细胞形式存在。当环境中的化合态氮源缺乏时,会沿藻丝以每10个营养细胞为间隔分裂出可以进行固氮作用的异形细胞,将氮气固定为化合态的氮源,并传输给临近的营养细胞,从而帮助整个藻丝渡过氮源匮乏的时期。因此,鱼腥蓝细菌是作为研究细胞分化良好的模式生物。2-酮戊二酸(2-oxogluatarate, 2-OG),来自高度保守的三羧酸循环途径,不仅是一个关键的代谢产物,同时也在许多细菌、植物甚至高等动物中,作为一种信号分子。在蓝细菌中,2-OG被证明是蓝细菌氮源缺乏的信号分子。而全局转录因子NtcA与信号传导蛋白被认为是2-OG的感应蛋白。NtcA属于Crp-Fnr转录因子家族,其典型的成员是结合cAMP的CAP蛋白。我们解析了鱼腥蓝细菌PCC 7120中NtcA的apo-form(1.90 (A|°))、结合2-OG(2.60 (A|°))和结合2-OG的不可代谢类似物—2,2-二氟代戊二酸(2.40 (A|°))的三个结构。三个结构都是同源二体的形式,每个亚基上有一个N端的效应分子结合结构域和一个C端的DNA结合结构域,两个结构域之间由一个长的螺旋(C螺旋)连接。2-OG结合在效应分子结合结构域的口袋中,这与CAP蛋白的cAMP结合位点相似,但是结合的具体方式不同。通过结构比较分析可以推导出2-OG结合引起NtcA可能的信号传递路径:2-OG的结合导致C螺旋之间形成更紧密的coiled-coil构象,从而使两个DNA结合螺旋(F螺旋)间的距离拉近以更适合DNA识别。cAMP的结合使CAP蛋白的激活状态发生从无到有的改变,而我们解析的结构则解释了为何NtcA在apo的状态也有DNA的结合能力,而2-OG的结合只是进一步提高这一结合能力。这些结果为2-OG调控的全局转录因子的功能提供了结构生物学的依据。PII是细菌、古细菌和植物中高度保守的信号传导蛋白。有一个大的柔性环状结构(T-loop),通过发生共价修饰或者结合不同的效应分子采取不同的构象以调节不同靶蛋白的功能。PipX是最早在单细胞蓝细菌Synechococcus PCC 7942中鉴定出的PII的一种结合蛋白,仅存在于蓝细菌中,并且PipX与NtcA也存在相互作用。PipX与PII和NtcA的结合是受2-OG浓度调节的,这使得PII间接调节了NtcA的转录活性。我们解析了PII–PipX复合物1.90 (A|°)的结构。PII同源三体通过三个T-loop与三个PipX结合。PII与之前报导的Synechococcus中的同源结构类似,由反向平行四个β折叠片的核心结构和侧面两个α螺旋组成。PipX则是一个新的结构,由五个扭曲的反向平行β折叠片和两个α螺旋组成,这与P_Ⅱ的结构很相似。每个P_Ⅱ亚基的T-loop像天线一样从核心结构伸出,以氢键介导与两个PipX分子之间的结合。此外,PipX的β片与的P_Ⅱ核心结构间的相互作用也为复合物的形成提供了一部分帮助。结构比较分析结果显示,PipX与2-OG在上的结合位点是重合的,并且与蓝细菌中P_Ⅱ的14个特征残基有交集。我们利用该复合物中PipX的结构与前面解析NtcA结合2-OG的结构模拟出NtcA–PipX复合物的结构,模型显示PipX利用相近的区域与P_Ⅱ和NtcA产生相互作用。这些结果为P_Ⅱ是如何通过PipX在2-OG的浓度依赖下调节NtcA的活性提供了结构生物学依据。本文的工作围绕鱼腥蓝细菌异形细胞分化早期调控蛋白的结构进行研究,为异形胞分化的调节机制提供了新的结构基础。更多还原

【Abstract】 Cyanobacteria are prokaryotes, also known as“blue-green algae”, which can execute photosynthesis. Some cyanobacteria can also fix nitrogen, such as the filamental cyanobacterium Anabaena, which live in the fresh water. When the nutrition is abundant, there are vegetative cells along the filament. While the envoriment is short of combined nitrogen, some vegetative cells will develop into heterocysts, usually every other 10 vegetative cells. These heterocysts can fix nitrogen, by transforming it into a combined form and transporting the fixed nitrogen to the neighbor vegetative cells, so that the whole filament can survive from the nitrogen starvation. These characteristics make Anabaena an excellent model for studying the cell differentiation.2-oxogluatarate (2-OG), a metabolite of the highly conserved Krebs cycle, not only plays a critical role in metabolism, but also constitutes a signaling molecule in a variety of organisms ranging from bacteria to plants and animals. In cyanobacteria, accumulation of 2-OG constitutes the signal of nitrogen starvation and NtcA, a global transcription factor, has been proposed to be a putative receptor of 2-OG. NtcA belongs to the Crp-Fnr family, which is featured by CAP (cAMP-activated catabolite activator protein). Here we present three crystal structures of NtcA from the cyanobacterium Anabaena sp. strain PCC 7120: the apo-form (1.90 (A|°)), and two ligand-bound forms in complex with either 2-OG (2.60 (A|°)) or its analogue 2,2-difluoropentanedioic acid (2.40 (A|°)). All structures assemble as homodimers, with each subunit composed of an N-terminal effector binding domain (EBD) and a C-terminal DNA binding domain (DBD) connected by a long helix (C-helix). The 2-OG binds to the EBD at a pocket similar to that used by cAMP in CAP (cAMP-activated catabolite activator protein), but with a different pattern. Comparative structural analysis reveals a putative signal transmission route upon 2-OG binding. A tighter coiled-coil conformation of the two C-helices induced by 2-OG is crucial to maintain the proper distance between the two F-helices for DNA recognition. While CAP adopts a transition from off to on state upon cAMP binding, our structural analysis explains well why NtcA can bind to DNA even in its apo form, how 2-OG just enhances the DNA-binding activity of NtcA. These findings provided the structural insights into the function of a global transcription factor regulated by 2-OG, a metabolite standing at a crossroad between carbon and nitrogen metabolisms. P_Ⅱproteins are highly conserved signal transducers in bacteria, archaea and plants. They have a large flexible loop (T-loop) that adopts different conformations after covalent modification or binding to different effectors, to regulate the functions of diverse protein partners. The P_Ⅱpartner PipX, first identified from Synechococcus sp. PCC 7942, exists uniquely in cyanobacteria. PipX also interacts with the cyanobacterial global nitrogen regulator NtcA. The mutually exclusive binding of P_Ⅱand NtcA by PipX in a 2-OG-dependent manner enables P_Ⅱto indirectly regulate the transcriptional activity of NtcA. We solved the crystal structure of the P_Ⅱ–PipX complex from the filamentous cyanobacterium Anabaena sp. PCC 7120 at 1.90 (A|°) resolution. A homotrimeric P_Ⅱcaptures three subunits of PipX through the T-loops. Similar to P_Ⅱfrom Synechococcus, the core structure consists of an antiparallelβ-sheet with fourβ-strands and twoα-helices at the lateral surface. PipX adopts a novel structure composed of five twisted antiparallelβ-strands and twoα-helices, which is reminiscent of the P_Ⅱstructure. The T-loop of each P_Ⅱsubunit extends from the core structure as an antenna that is stabilized at the cleft between two PipX monomers via hydrogen bonds. In addition, the interfaces between theβ-sheets of PipX and P_Ⅱcore structures partially contribute to complex formation. Comparative structural analysis indicated that PipX and 2-OG share a common binding site that overlaps with the 14 signature residues of cyanobacterial P_Ⅱproteins. Our structure of PipX and the recently solved NtcA structure enabled us to propose a putative model for the NtcA–PipX complex. Taken together, these findings provide structural insights into how P_Ⅱregulates the transcriptional activity of NtcA via PipX upon accumulation of the metabolite 2-OG.This study fucosed on the proteins involved in the early stage of heterocyst development and these output results provided structural insights for the regulatory mechanism of heterocyst development.更多还原