Study On The Structure And Funtion Of A Novel Ubiquitin Modification And Ligase Sdea In Legionella Pneumophila

Legionella pneumophila is widely distributed in nature,and its survival and replication are achieved by secreting toxic effector proteins into host cells,altering host cell functions,and inhibiting endocytic pathways of host cells.Legionella pneumophila can infect the lungs and cause pneumonia,posing a great danger to human health.The reproduction of Legionella pneumophila in host cells is dependent on its Dot/Icm secretion system,which can transfer hundreds of toxic effectors to host cells.This paper studies the molecular mechanism of specific ubiquitination mediated by one of the toxic effector proteins SdeA,focusing on the structural biology of proteins involved in this ubiquitination process,and structural biology studies of complexes formed by SdeA and secretory system proteins DotL,IcmS and IcmW.In this thesis,molecular clones of different lengths of SdeA and different labels of ubiquitin(Ub)were successfully constructed and successfully expressed in E.coli.The purity and homogeneity of the purified protein met the crystallization requirements.Through the co-crystallization experiment,the crystals of SdeA and Ub complexes were successfully obtained.After X-ray diffraction,the crystals received a resolution of 3.1A,and the three-dimensional structure of the effect protein SdeA231-1190aa and Ub complexes was analyzed.The crystal structure demonstrates the combination and interaction of the SdeA231-1190aa and Ub at the molecular level,which lays an important structural foundation for the special ubiquitination mediated by SdeA.Another part of this paper is about the purification and crystallization of the effector protein SdeA and IcmS/IcmW/DotL complex.We first constructed molecular clones of IcmS,IcmW,DotL and IcmS+IcmW,and successfully expressed them in E.coli,and obtained IcmS/IcmW binary complex,IcmS/IcmW/DotL ternary complex and SdeA/IcmS/IcmW/DotL quaternary complex,after finding a lot of crystallization conditions,has successfully obtained the protein crystal of the quaternary complex,data collection is in progress.