Structural Insights Into Effector Protein Lem23/lpg2406 From Legionella Pneumophila

Legionella pneumophila(L.pneumophila),the causative agent of the Legionnaires' disease,injects nearly 330 virulence factors(commonly known as effectors)into host cell through its unique type IV B(Dot/Icm)secretion system to escape the host immune surveillance and promote its proliferation.Lem23(also known as lpg2406)is one of identified L.pneumophila effectors delivered into host cell through the Dot/Icm secretion system that remains uncharacterized.In order to understand the role of Lem23 during the invasion of host cell and seek its intracellular targets,we solved the three-dimensional structure of Lem23 and performed initial analysis of its functions.(1)Analysis of Lem23 structure.Primers were designed to amplify Lem23 gene,which was then cloned into p MCSG7 plasmid to form a recombinant vector,and IPTG was used to induce protein expression in E.coli Rosetta(DE3)expression system.Lem23 was successively purified by Ni-NTA affinity chromatography,ion-exchange chromatography and size-exclusion chromatography.Commercial crystallization kits were used for preliminary crystallization screening,and crystals with high diffraction were obtained after optimizing the hits.We collected diffraction data,then the three-dimensional structure of Lem23 was analyzed using Single-wavelength Anomalous Diffraction,and finaly the model was built and corrected by Coot and REFMAC servors.(2)Functional analysis based on the structure of Lem23.We used molecular interaction detection techniques such as liquid chromatography-mass spectrometry(LC-MS),isothermal titration calorimetry(ITC)and microscale thermophoresis(MST)to study the affinities of Lem23 for physiologically relevant phosphate-containing small molecules(i.e.ATP,ADP,AMP,AMPPNP,NADH,NADPH,dCTP and dGTP)and to determine whether RIN4 was the host target of Lem23.(3)The possibility that Lem23 may form a functional complex with effectors lpg2405,lpg2404,lpg2407 belonging to the same operon was also investigated using molecular interaction detection techniques.The three-dimensional structure of Lem23 exhibits a bilobed architecture,with the lower lobe containing a ?-sheet fold arranged into a palm-like curved surface and the upper lobe forming a globular shape comprising a predominantly ?-helical FIC(filamentation induced by cyclic adenosine monophosphate moiety)-like domain.Structural comparison revealed that Lem23 bears similarity to L.pneumophila effectors AnkX,Homo sapiens FICD and Pseudomonas syringae effector AvrB,all of which are members of Fido domain superfamily,thus providing a valuable clue for functional characterization of Lem23.A conserved binding pocket was observed in Lem23,which considering the results of structural comparison was hypothesized to bind phosphate-containing small molecules.Although our molecular interaction experiments indicated that Lem23 has no direct interaction with the tested molecules(ATP,ADP,AMP,AMPPNP,NADH,NADPH,dCTP and dGTP),the results of molecular dynamics study between Lem23 and ADP suggested that Lem23 may need to undergo conformational activation before being able to bind small molecules.Furthermore,RIN4(RPM1-interacting protein 4,RIN4)was found to be a potential host target of Lem23,and effector proteins located within the same operon as Lem23 were not observed to assemble into functional complexes with Lem23.In conclusion,although the three-dimensional structure of Lem23 has been analyzed and its potential host target has been identified,further studies are required to determine specific functions of Lem23.