Structural And Functional Of The Legionella Pneumophila Effector Lgt2

Glycosylation modification is a post-translational modification of proteins catalyzed by glycosyltransferases,which exists widely in organisms,and is closely related to the virulence of pathogenic bacteria.Legionella pneumophila is an intracellular pathogen that transports nearly 300 effector proteins to host cells through its IVB-type secretion system,which can mediate diverse post-translational modifications of proteins to interfere with host cellular processes.Among them,effectors such as Lgt family(including Lgt1-3),Sid I,Set A and Ltp M mediate host protein glycosylation and hijack host protein translation and vesicle transport,thus benefitting to intercellular replication and proliferation of Legionella pneumophila.It has been reported that the Lgt family glycosylated the transcription elongation factor e EF1 A,thus inhibiting the host protein synthesis.Lgt family co-regulates host m TORC1 by cooperation with the Sid E family,thereby participating in regulation of host nutrient and metabolism.In addition,Lgt family inhibit the endoplasmic reticulum stress by inhibiting splice of the regulatory factor XBP1.In addition,the Lgt family are differentially regulated during the process of Legionella infection,which is closely related to Legionella infection and the release of Legionella at the later stage of infection.Lgt2 is special in the Lgt family,for example,Lgt2 has one more domain at N-terminal than Lgt1 and less domain at C-terminal than Lgt3 by sequence analysis.In addition,only Lgt2 has auto-glycosylation and its activity consensus substrate e EF1 A is low in the Lgt family,which implies that Lgt2 may have different catalytic mechanisms.In the Lgt family,only the structure of Lgt1 has been solved,the structures of Lgt2 and Lgt3 have not been reported.Therefore,this study focuses on the glycosyltransferase Lgt2 of Legionella pneumophila,analyzed the structure of Lgt2 and its complex with UPD-Glucose by structural biology,in order to clarify the catalytic mechanism of Lgt2.In this study,the apo form structure of Lgt2(2.27 (?))and the binary complex structure of Lgt2 and UDP-Glucose(2.46 (?))were solved by X-ray.Lgt2 belongs to the GT-A fold type glycosyltransferase,in which the GT-I domain contains two Rossmann-like domains(α11-β3-α12 and α13-β4-α14)and the catalytic center DXD motif by structural analysis.In vitro,we combined Lgt2 binary complex structure,glycosyltransferase activity assay and ITC assay to determine its binding pocket and active center.Among them,amino acids D320 and A339 are involved in the binding of UDP-Glucose;amino acids N384,N602,D338 and D340 are related to the transglycosylation activity of Lgt2.The retaining mechanism is used to catalytic the UDP-Glucose and Lgt2 has a different recognition mechanism for substrate between Lgt1;in vivo,we found that NTD I domain is required for intracellular localization.In addition,we found that Lgt2 inhibited the expression of the receptor protein ATF6 and the ER stress effectors ATF4,Bi P and CHOP,thereby inhibiting the unfolded protein response.Our study not only revealed the mechanism of Lgt2 catalyzes UDP-Glucose,but also further explored its cellular biological function,which provided a basis for further study of Legionella pneumophila glycosyltransferase.