【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， 2021， 博士

【摘要】 金黄色葡萄球菌作为一种常见的人体共生致病菌,它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,一种新型的核苷酸磷酸酶Sa1684被鉴定出来,它的缺失可以改变与核苷代谢、糖酵解途径、发酵途径相关的多种基因的表达,还不同程度地影响多种重要转录调节因子(如agr、sarX和sarZ)和毒力因子(如溶血素、核酸酶)的表达。此外,Sa1684的缺失可以在不影响金葡菌正常生长的情况下,显著降低其菌落的传播能力和对蚕的侵染能力。这使得Sa1684成为了一种极好的潜在的金黄色葡萄球菌感染药物的设计靶点。为了真正了解Sa1684的催化机制以及Sa1684蛋白与其底物之间的相互作用关系,需要获得Sa1684及其与底物复合物的结构。我们通过体外重组表达获得了Sa1684蛋白,并鉴定了它具有NDP和NTP的水解能力。然后利用X射线晶体衍射的方法解析了 apo-Sa1684,Sa1684-ATPγS,Sa1684-GDPβS和Sa1684-GTPγS的三维结构。通过三种底物复合物的结构比对与突变体酶促动力学的分析,验证了磷酸键水解中心的结构组成,并且提出了可能的催化水解的机制。根据该水解中心磷酸盐的结合位置以及UHPLC的测定结果,我们确定了Sa1684的底物水解过程。通过底物结合相关的氨基酸的突变与其酶促动力学的测定,鉴定了 Asn45在GTP选择性上的重要作用。此外,通过比较不同蛋白的底物结合模式,我们发现Sa1684具有更加开放的底物结合口袋;并且保守的DUF402结构域是形成催化水解中心的基础结构。虽然Sa1684可以水解多种底物,但是其对GTP的特异性水解可能与其生理功能有关,对此我们推测了Sa1684蛋白参与生理调节的可能途径。在细胞分裂过程中,姐妹染色体高保真的分离是极其重要的过程。这一过程中的任何的异常或者错误都会给细胞带来灾难性的破坏,而这一过程合理有序进行的依托是极其复杂的蛋白质复合物机器以及激酶调控网络,包括着丝粒、动粒蛋白复合物、微管纤维丝以及一系列的激酶。动粒作为连接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。截止目前,100多个动粒组分被揭示,包括大约30个核心成分,如组成性着丝点相关网络CCAN和KMN网络。酿酒酵母作为经典的模式生物,是用来研究动粒的理想模型。在酿酒酵母中,Ctf3作为Ctf19CCAN合物的重要组成部分,它可以与Mcm16,Mcm22,Cnn1和Wip1形成一个五元复合物,在动粒机器的组装以及细胞分裂的过程中发挥重要作用。但是目前为止,Ctf3复合物的研究只停留在了生理作用层面,其具体的组装模式以及各个亚基之间的相互作用机制尚不清楚。我们尝试对Ctf3复合物进行了体外重组表达以及三维结构的解析。实验之初,我们通过体外相互作用实验初步鉴定了该复合物可能的组装结构以及与部分其他动粒蛋白复合物之间的相互作用关系。实验结果表明,Mcm16-Mcm22亚复合物无法在Ctf3不参与的条件下与Cnnl-Wip亚复合物相互作用;且体外相互作用实验证明,Ctf3五元复合物无法在体外与Chl4-Iml3复合物进行相互作用组装。为了获得Ctf3复合物的三维结构,我们分别采用了 X射线晶体衍射以及Cryo-EM的方法对其进行结构解析。通过X射线晶体衍射的方法我们获得了 4 A左右分辨率的Ctf3五元复合物晶体,但是由于分辨率以及各向异性的原因无法对其结构进行解析。此外,通过冷冻电镜的方法,最终也只获得了 8 A左右的模型。为了提高Ctf3复合物晶体衍射的分辨率,我们分别尝试了抗体的筛选,胰蛋白酶酶切实验,cross-link实验,蛋白甲基化实验以及重组表达可能具有相互作用的动粒蛋白亚复合物来稳定Ctf3复合物等方法,但是也都没有得到预期的结果。更多还原

【Abstract】 Staphylococcus aureus,as a common human symbiotic pathogen,cause a series of human infections by regulating the release of virulence factors.Recently,a novel nucleotide phosphatase(Sa1684)has been identified.Its deletion alter the expression of several genes related to nucleoside metabolism,glycolysis pathway,and fermentation pathway,and also affect the expression of several important transcriptional regulatory factors(such as agr,SarX,and SarZ)and virulence factors(such as hemolysin and nuclease)to varying extent.In addition,the absence of Sa1684 significantly reduces the spread ability of colony and reduced the infection ability to silkworms without affecting the normal growth of S.aureus which makes Sa1684 an excellent potential drug design target for Staphylococcus aureus infections.Therefore,to truly understand the catalytic mechanism of Sa1684 and the substrates interaction network,it is necessary to obtain the apo structure of Sa1684 and the substrate-complexed structures of Sa1684.We obtained the Sa1684 protein by vitro recombinant expression,and identified that Sa1684 has the hydrolytic ability to NDPs(nucleoside di-phosphate)and NTPs(nucleoside tri-phosphate).Then,the three-dimensional structures of apo-Sa1684,Sa1684-ATPyS,Sa1684-GDPpS and Sa1684-GTPγS were solved by X-ray crystal diffraction.The structural composition of the hydrolyzing center was verified by structural analysis and the mutants’ catalytic kinetics measuredments.Furthermore,the possible catalytic hydrolysis mechanism was given.According to the binding position of terminal phosphate in the hydrolysis center and the results of UHPLC,we determined the hydrolysis process of NTPs.By measuring the catalytic kinetics of the substrate-related mutants,the important role of Asn45 in GTP selectivity was determined.In addition,by comparing the substrate binding patterns of different proteins(Sa1684,Sc4828 and SfFomD),we found that Sa1684 had a more open substrate binding pockets.The conserved DUF402 domain was identified as the basal catalytic hydrolysis center.Based on the high specificity of Sa1684 to GTP,we speculated the possible pathway of Sa1684 protein involved in physiological regulation.High fidelity separation of sister chromosomes is an important process during cell division.Any aberration or error in this process can cause catastrophic damage to the cells,and this process runs stablely on the basis of complicated protein interaction network and kinases regulations,including centromere,kinetochore complexes,microtubule filaments,and a series of kinases.As the intermediate bridge between centromere and microtubules,the kinetochore plays an indispensable role in the process of cell division.Up to now,more than 100 kinetochore components have been revealed,including about 30 core components,such as the constituent centromere-related network CCAN and KMN networks.Saccharomyces cerevisiae,which is a classic model organism,is an ideal model for the study of kinetochore.In Saccharomyces cerevisiae,Ctf3,as an important component of the Ctf19CCAN complex,forms a pentabasic complex with Mcm 16,Mcm22,Cnn1 and Wip1,which plays an important role in the assembly of kinetochore.However,so far,Ctf3 complex has only been studied at the physiological level,and its specific assembly mode and the interaction mechanism among the subunits remain unclear.We attempted to obtain the Ctf3 complex in vitro and solve its three-dimensional structure.At the beginning of the experiment,we preliminarily identified the possiblely assembly model of the complex and its interaction with some other kinetochore protein complexes by in vitro interaction experiments.The results showed that the Mcm16-Mcm22 subcomplex could not interact with Cnnl-Wip1 subcomplex in the absence of Ctf3 protein.Moreover,vitro interaction experiments showed that Ctf3 complex could not be assembled with Chl4-Iml3 complex in vitro.In order to obtain the three-dimensional structure of Ctf3 complex,X-ray crystal diffraction and Cryo-EM were used to solve the structure of Ctf3 complex.We obtained Ctf3 complex crystals with a resolution of about 4 A by X-ray crystal diffraction.However,due to the low resolution and anisotropy,the structure of Ctf3 can not be solved.In addition,a model with about 8 A resolution was obtained by cryo-electron microscopy.In order to improve the crystal diffraction resolution of Ctf3 complex,we tried some methods,such as antibody screening,trypsin digestion test,cross-link test,protein methylation test and recombinant expression of kinetochore protein subcomplex that may interact with each other to stabilize Ctf3 complex,but none of the expected results have been obtained.更多还原