Molecular Mechanism Of The Dual Catalytic Activity Of Legionella Pneumophila Deubiquitinase LotA

Ubiquitylation is an important post-translational modification（PTM）in eukaryotes,which is involved in regulating almost every link of the eukaryotic cell life cycle,such as cell division,DNA damage repair and protein synthesis.In addition,the ubiquitination system has an important function in the defense against pathogenic infection.Ubiquitination can sense invading bacteria and kill pathogens through proteasome pathway or lysosome,autophagy and other pathways.Recent studies have shown that the effector proteins delivered by many pathogens to the host through specific types of secretion systems function as ubiquitinases,which can lead to disease by disrupting the host’s ubiquitination process.Several effector proteins secreted by Legionella pneumophila have E3ubiquitin ligase or deubiquitinase functions,among which the effector protein LotA is the first OTU family DUBs identified.It has two DUB domains,DUB1 is dependent on Cys-13 and specifically recognizes K6-linked ubiquitin chains;DUB2 is dependent on Cys-303 to cleave polyubiquitin chains.However,the molecular mechanism of the dual catalytic activity of LotA remains to be studied.（1）In this paper,LotA was overexpressed in E.coli expression system first,and by changing the expression vector,we tried to construct different truncations,and purified to obtain stable LotA protein.（2）LotA and Ub-PA can form a covalent complex after in vitro incubation;in cell experiments,it also has a good inhibitory effect on host ubiquitination modification in 293T cells,and DUB2 has a better cleavage effect on polyubiquitin.（3）In order to further explore the recognition and cleavage mechanism of ubiquitin by LotA,the crystal structure of LotA_（1-542）and Ub-PA complex was solved by X-ray crystallography（X-ray）with a resolution of 2.64?.（4）Surprisingly,the LotA DUB1 domain binds specifically to two Ub-PA molecules（S1 and S1’site）,and we found a K6 distance of 4.9?between the C-terminus of the S1 Ub and the S1’Ub,mimicking the K6-linked di-Ub.Based on the structural analysis and the cleavage experiments of different types of di-Ub by LotA,the structural basis of the high specificity of LotA DUB1 for K6-linked di-Ub was revealed.（5）In DUB2,no binding to Ub-PA was found from the solved structure,but through molecular simulation and combined with biochemical experiments,it was verified that D410 located on the extra helix of LotA DUB2 is critical for its specific recognition of polyubiquitin.In conclusion,the results of this study revealed the first molecular mechanism for the recognition of K6-linked ubiquitin chain OTU family DUBs,providing structural evidence for the study of the specific recognition mechanism of OTU family DUBs.However,the biological function of LotA in the process of Legionella infection still needs further research.