【作者】 王振华；

【导师】 滕脉坤； 李旭；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2022， 博士

【摘要】 本论文主要包含两个课题的研究工作。第一部分为金黄色葡萄球菌中核苷磷酸酶Sa1684的结构与功能研究,第二部分为酿酒酵母动粒蛋白复合物Ctf3的组装机制研究。金黄色葡萄球菌作为一种常见的人体共生致病菌,可以通过调节毒力因子的释放来造成一系列的人体感染。最近,人们鉴定出一种新型的金黄色葡萄球菌核苷酸磷酸酶Sa1684,它的缺失可不同程度地影响多种重要转录调节因子和毒力因子的表达,在金葡菌的侵染过程中发挥重要作用。作为一个在金葡菌中初步被鉴定的功能性核苷磷酸酶,对其酶学作用机制及结构与功能关系的了解不足。在论文的第一篇,我们通过体外重组表达与纯化,获得了稳定且均一的蛋白样品;利用X射线晶体学方法解析了Sa1684及其与三种底物的复合物结构;通过结构分析并结合相关的酶促动力学研究,揭示了Sa1684的底物水解机制以及底物(GTP)偏好性的结构基础。综合实验结果和已有的文献报道,我们推测Sa1684蛋白通过控制细胞内GTP的浓度来调节CodY的活性,从而影响转录因子和毒力因子的表达。动粒作为着丝粒和微管的中间桥梁,在细胞分裂过程中起着不可或缺的作用。Ctf3p是Ctf19CCAN动粒复合物的重要组成部分,它可以与Mcm16,Mcm22,Cnn1和Wip1形成一个稳定的五元复合物,Ctf3p的缺失会严重影响细胞分裂的进行。到目前为止,Ctf19CCAN复合物中除了Ctf3复合物以外,其他的各个亚基(亚复合物)的结构都得到了一定的解析。为了填补这最后一块空缺,Ctf3复合物的结构解析则成了较为热门的研究方向。在本论文的第二篇中,我们对Ctf3复合物进行了体外重组表达、X射线晶体衍射、冷冻电镜和pull-down相互作用等研究。确定了Ctf3复合物中各组分之间的相互作用关系;为了获得稳定的Ctf3复合物,我们开展了抗体的筛选、胰蛋白酶酶切、cross-link和蛋白甲基化等多种方法的尝试。最终获得了 4 ?左右分辨率的Ctf3五元复合物晶体,同时还利用冷冻电镜的方法获得了 8 ?左右的模型。更多还原

【Abstract】 This thesis mainly includes two research projects.The first part is about the structure and function of nucleoside phosphatase Sa1684 in Staphylococcus aureus.The second part is about the assembly mechanism of Ctf3 complex in S.cerevisiae.Staphylococcus aureus,as a common human symbiotic pathogen,can cause a series of human infections by regulating the release of virulence factors.Recently,a novel nucleotide phosphatase,Sa1684,has been identified.Its deletion can affect the expression of many important transcriptional regulatory factors and virulence factors to varying degrees,and it plays an important role in the infection process of S.aureus.As a novel nucleoside phosphatase dentified in S.aureus,the mechanism of enzymatic action and the relationship between structure and function of the nucleoside phosphatase are not well understood.In the first part of this paper,we made Sa1684 as the research target,and obtained stable and highly purified protein samples through recombinant expression and purification.We solved the structures of Sa1684 and its complex with three substrates by X-ray crystal diffraction.The structures of Sa1684 was analyzed and the enzymatic kinetics study was carried out to reveal the substrate hydrolysis mechanism and the structural basis of GTP preference.Based on the experimental results and existing literature reports,we speculated that Sa1684 regulates the activity of the CodY by controlling the concentration of intracellular GTP to affect the expression of transcription factors and virulence factors.Kinetochores,as the bridge between centromere and microtubule,play an indispensable role in cell division.Ctf3p is an important part of the Ctf19CCAN kinetochore complex,which can form a stable five-subunits complex with Mcm 16,Mcm22,Cnn1 and Wip1.Its deletion will seriously affect the progress of cell division.Till now,except Ctf3 complex,the structures of other subunits or subcomplexes in Ctfl9CCAN complex have been elucidated to some extent.In order to fil1 this last gap,the structural analysis of Ctf3 complex has become a popular research direction.In the second part of this paper,Ctf3 complex was studied by in vitro recombinant expression,X-ray crystal diffraction,cryo-electron microscopy and pull-down interaction.The interactions among components of Ctf3 complex was determined.In order to obtain stable Ctf3 complex,we have carried out antibody screening,trypsin digestion,cross-link and protein methylation assays.Finally,Ctf3 five-subunits complex crystals with a resolution of about 4 ? was obtained,and a model about 8 ? resolution was also obtained by cryo-EM.更多还原