The Catalytic Mechanism And Structural Basis Of The Effector Protein MvcA Of Legionella Pneumophila In Regulating The Host Ubiquitination System

Legionella pneumophila,as an aerobic gram-negative bacillus,facultatively parasitizes human monocytes and macrophages,and can cause acquired atypical pneumonia.When infecting the host,Legionella pneumophila can secrete more than 300 kinds of effectors into the host cytoplasm through its unique Dot/Icm（Defective organelle trafficking/Intracellular multiplication）IVB secretion system.The functions of these effectors include the formation of Infect vesicle LCV（Legionella-containing Vacuole）,inhibit host immune signal transduction,"hijack" important regulatory proteins in host cells,and induce host cell apoptosis and lysis.In recent years,there have been increasing cases of Legionella pneumophila infections reported at home and abroad,and more than 90% of Legionella strains are resistant to antibiotics.Therefore,we focus on studying the mechanism of Legionella pneumophila effector protein,hoping to use this as an entry point to develop targeted new drugs.Some studies have shown that effector proteins of Legionella pneumophila can regulate and utilize the host ubiquitination system in different ways.Recently,two articles have reported the function of the effector MavC（Lpg2147）of Legionella pneumophila.MavC can catalyze the monoubiquitination of E2 ubiquitin conjugating enzyme UBE2 N through transglutaminase activity.This catalytic process prevents UBE2 N from participating in the formation of the K63 polyubiquitination chain,thereby inhibiting the activation of the NF-κB signaling pathway.In addition,another effector of Legionella pneumophila,MvcA（Lpg2148）,can specifically reverse the ubiquitination process catalyzed by MavC,thereby precisely regulating host signals during infection.Therefore,this study intends to take the Legionella pneumophila effector protein MvcA as the primary research object of this topic,combined with biochemistry,structural biology and other means,to clarify from the molecular biology level that MvcA plays a deubiquitinated modification effect to regulate the corresponding cell process of the host Specific mechanism.In this paper,the E.coli expression system was used to express and purify the four proteins of MvcA,MavC,UBE2 N and Ub,and the MvcA-UBE2N-Ub ternary complex was prepared,and the ternary complex crystal with a resolution of 2.45 ? was successfully obtained.Analyzing the structure of the complex,it was found that UBE2 N and Ub were placed on both sides of the central domain of MvcA,and the isopeptide bond formed between UBE2 N and Ub was tightly bound to the catalytic triplet enzyme center of MvcA.Among them,the insertion domain of MvcA participates in the recognition and binding of UBE2N-Ub（especially the UBE2 N part）.Mutations of the key amino acids that interact between UBE2 N and Ub and MvcA revealed that the mutations of Arg140,Asp208,Asn88,Glu137 and Lys177 amino acid sites will affect the deubiquitination activity of MvcA.In summary,this paper studies and analyzes the structure of the complex of MvcA and UBE2N-Ub,and clarifies the molecular mechanism of the effector protein MvcA catalyzing the deubiquitination of UBE2N-Ub from the perspective of structural biology.