绿脓杆菌RNaseE抑制因子RraA和鞭毛hook组装蛋白FlgD的结构和功能研究

发布时间：2018-05-02 12:14

本文选题：RraA + 晶体结构　；参考：《重庆医科大学》2010年博士论文

【摘要】： 第一部分RNase E抑制因子RraA的结构和功能研究 RNase E(ribonuclease E,核糖核酸酶E)是广泛地存在于植物和细菌细胞中,是参与众多功能RNA成熟和mRNA降解代谢过程中的限速酶。以RNase E为组装支架,PNPase(核苷酸磷酸化酶)三聚体、RhlB(RNA酶解螺旋酶B)单体以及enolase(烯醇化酶)二聚体的相互作用结合共同组成mRNA降解体功能复合物,共同协调完成对mRNA的降解过程。RraA是至今为止发现的对RNase E起抑制作用的调控因子,已有的研究证实RraA可与降解体复合物其它几种组分PNPase、RhlB和enolase在RNase E的C端非催化区域结合位点的竞争结合,并降低该几种组分在RNA降解体复合物中含量达到其抑制作用。蛋白质的结构特征决定其功能的行使。为了进一步了解RraA对降解体功能复合物在mRNA降解处理过程中的作用,我们选取临床上常见的机会致病菌-绿脓杆菌(Pseudomonas aeruginosa)为研究对象;克隆、表达、纯化并利用气相扩散法结晶获得了绿脓杆菌RraA(PaRraA)晶体,使用in house X射线光源收集了一套分辨率为2.0?的衍射数据;以大肠杆菌(E.coli)RraA(PDB code 1q5x)为结构搭建模型,利用分子置换法(Molecular Replacement,MR)获得了PaRraA晶体三维空间结构模型(Rfactor =20.8%,Rfree=24.2%,PDB code 3C8O)。通过结构分析发现:PaRraA与已知的其它物种来源的RraA分子结构相似,为结构保守的蛋白质。利用激光光散射实验和凝胶层析实验本研究首次证实:PaRraA在溶液中为六聚体形式,符合晶体中的“两个三聚体形成的二聚体”这一分子堆积方式;通过蛋白质结构和序列分析发现:PaRraA六聚体分子含有的六个带电荷区域,提示其可能参与该分子同降解体其它组分-PNPase、RhlB和enolase在RNase E的C端非催化区域的竞争结合,从而调节降解体的降解活性,这为进一步理解该分子对体内RNA降解机制提供了理论依据。第二部分细菌鞭毛hook组装蛋白FlgD的结构和功能研究鞭毛是致病菌重要的毒力因子,参与细菌与宿主的相互作用。细菌的鞭毛是由超过50个基因编码的蛋白质进行组装和调控。鞭毛主要由基体(basal body)、鞭毛钩(hook)和鞭毛丝(filament)三个部分组成。其中,鞭毛沟(hook)象铰链一样联系鞭毛(filament)和细胞膜上的鞭毛基体(basal body),由hook蛋白质FlgE组装而成。FlgD是鞭毛hook组装过程中重要的支架分子(scaffolding protein);在未成熟的鞭毛hook组装过程中,FlgD形成的临时“帽子”结构亦可以阻挡hook蛋白FlgE的分泌泄露;另外,FlgD与FliK能共同调控FlgE聚合方式,使hook的长度在一定范围内。已有的研究证实:FlgD仅存在于大肠杆菌和沙门氏菌鞭毛hook的组装期,是鞭毛hook结构组装过程中的调控蛋白。迄今为止,关于FlgD蛋白分子的结构信息非常局限,仅有植物致病菌Xanthomonas campestris来源的FlgD分子结构得以解析,但仅有C端约130个氨基酸区域晶体结构信息(XcFlgD; PDB code 3c12)。尚未获得参与hook组装过程中起重要作用的N端结构信息。为了进一步了解该分子在鞭毛组装、调控等过程中的生物学作用,我们以临床常见的机会致病菌-绿脓杆菌为研究对象,通过flgd的克隆、表达、纯化、结晶,并对FlgD晶体进行X射线晶体衍射获得了母体衍射数据;利用在上海同步辐射收集的Se-Met多波长反常散射数据,应用XPREP寻找Se重原子的坐标,利用Solve解析相位和Resolve进行相位优化和模型搭建,然后运用Coot和Refmac进行结构修正,完成了PaFlgD分辨率为2.4?的三维结构的解析(PDB code 3IOA)。实验获得了C端131个氨基酸蛋白质分子空间结构,与降解实验获得的14.5kDa的片段相吻合。获得的PaFlgD分子结构为两个较独立的结构域:Tudor结构域和Fn-Ⅲ结构域杂交组合而形成的结构,其中β折叠(β-strand)含量非常丰富,相关的结构和功能关系研究正在进行中。
[Abstract]:The first part is about the structure and function of RNase E inhibitor RraA.
RNase E (ribonuclease E, ribonuclease E) is widely existed in plant and bacterial cells, and is the speed limiting enzyme involved in many functional RNA maturation and mRNA degradation and metabolism. RNase E is the assembly scaffold, PNPase (nucleotides phosphorylase) trimer, RhlB (RNA enzymolysis helicase) monomer and two polymer (enolase). The role of the combination of mRNA descending functional complex and co ordination to complete the degradation of mRNA.RraA is a regulatory factor that has been found so far on the inhibition of RNase E. The previous studies have confirmed that RraA can be associated with the other components of the descending complex, PNPase, RhlB and enolase in the RNase E C terminal non catalytic regional binding sites. Competitive combination can reduce the content of these components in RNA degradation complex and achieve its inhibitory effect.
The structural characteristics of protein determine the exercise of its function. In order to further understand the role of RraA in the degradation process of mRNA, we select the common opportunistic pathogenic bacteria, Pseudomonas aeruginosa (Pseudomonas aeruginosa), as the research object; clone, express, purify and use the crystallization of gas phase diffusion to obtain green. Pseudomonas RraA (PaRraA) crystal, using in house X ray light source, collected a set of diffraction data with a resolution of 2?, and the structure model was built with E.coli RraA (PDB code 1q5x), and the three-dimensional spatial structure model of the crystal was obtained by molecular replacement. O). Through structural analysis, it is found that PaRraA is similar to the known RraA molecular structure of other known species and is a structurally conservative protein. It is first confirmed by laser light scattering experiment and gel chromatography experiment that PaRraA is in the form of six polymer in solution, conforming to the "two polymer" of "two trimers" in the crystal. Through protein structure and sequence analysis, it is found that the PaRraA six polymer contains six zone of charge with charge, suggesting that it may participate in the competition of -PNPase, RhlB and enolase in the non catalytic region of C end of RNase E, thus regulating the degradation activity of the descending body, which is a further understanding of the molecule. The mechanism of RNA degradation in the body provides a theoretical basis.
The second part is about the structure and function of bacterial flagellum hook assembly protein FlgD.
Flagellum is an important virulence factor of pathogenic bacteria and participates in the interaction between bacteria and host. The flagellum of bacteria is assembled and regulated by more than 50 genes encoding proteins. Flagellum is mainly composed of three parts, basal body, flagellum hook (hook) and flagellum (filament). Among them, flagellum (hook) is linked to flagella like hinges (FILA). Ment) and the flagellum (basal body) on the cell membrane, which are assembled by hook protein FlgE, are the important scaffolding molecules (scaffolding protein) in the hook assembly process of flagellum. In the process of the immature flagellum hook assembly, the temporary "hat" structure formed by FlgD can block the secretion leakage of the hook protein. The FlgE polymerization can be regulated together to make the length of hook within a certain range. Previous studies have confirmed that FlgD exists only in the assembly period of the flagellate hook of Escherichia coli and Salmonella, and is a regulatory protein in the process of hook structure assembly of flagellum.
So far, the structure information about FlgD protein molecules is very limited, only the FlgD molecular structure of the plant pathogenic bacteria Xanthomonas campestris is analyzed, but only the crystal structure information (XcFlgD; PDB code 3c12) of about 130 amino acid regions of the C end has not been obtained. In order to further understand the biological function of the molecule in the process of flagellum assembly and regulation, we use the common opportunistic pathogenic bacteria - Pseudomonas aeruginosa as the research object, through the cloning, expression, purification and crystallization of flgd, and the X ray diffraction of the FlgD crystal to obtain the matrix diffraction data; and use the Se-Me collected in Shanghai synchrotron radiation. T multi wavelength anomalous scattering data, using XPREP to find the coordinates of Se heavy atoms, using Solve analytical phase and Resolve for phase optimization and model construction, and then using Coot and Refmac to modify the structure, complete the resolution of the three-dimensional structure of PaFlgD with a resolution of 2.4? (PDB code 3IOA). The experiment obtained the 131 amino acid protein molecules on the C end. The spatial structure is consistent with the 14.5kDa fragments obtained by the degradation experiment. The PaFlgD molecular structure obtained is two more independent domains: the structure of the hybrid combination of the Tudor domain and the Fn- III domain, in which the content of beta folds (beta -strand) is very rich, and the related structural and functional relationships are in progress.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R378

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