Structural Basis Of The Legionella Pneumophila Effector Protein MavL

Legionella pneumophila,an intracellularly parasitic Gram-negative bacterium,can use its Type IVB secretion system（T4BSS）to deliver effector proteins that are involved in the regulation of host life activities and thus cause severe human pneumonia disease.After analyzing Legionella pneumophila effector proteins,researchers found that Legionella may have multiple effector proteins that use ADP-ribosylation to regulate host substrate proteins.ADP-ribosylation modification in cells is a dynamic process that includes ADP-ribosylation modification and de-modification,and the enzymes that perform these two processes are ADP-ribosyl transferases（ARTs）and ADP-ribosyl hydrolases（ARHs）.The ADP-ribosylation modification of Legionella effector proteins should also be a complete dynamic process,but most of the reports so far have been on ARTs in Legionella,and ARHs have been rarely reported.Encouragingly,we identified a new Legionella pneumophila ARH-MavL（Lpg2526）in our experiment.In this paper,we will use the Legionella pneumophila effector protein MavL as the object of study,and use molecular biology and structural biology techniques to elucidate the ADP-ribosyl hydrolase activity of MavL and its catalytic process at the molecular level,in an attempt to complement mechanism of host life activity regulation by Legionella using ADP ribosylation modification,and also provide a reference for the study of the catalytic mechanism of ARHs in Legionella.Through bioinformatics analysis of effector proteins of unknown function in Legionella pneumophila,we found that MavL may have the catalytic structural domain of Macro Domain family protein PARG（ADP ribosyl hydrolase protein）,and thus hypothesized that it may be an ADP ribosyl hydrolase,and with this we strated our experiment.（1）we confirmed in vitro by molecular biology that MavL does possess the properties of Macro Domain family proteins:it has in vitro cleavage activity for two reported proteins modified by ADP ribosylation in Legionella pneumophila（ADPr-Ub and ADPr-ANT1）,indicating that it does possess ADP ribosyl hydrolase activity.However,when we analyzed MavL with PARG for sequence conservation,we found that MavL does not possess the-GGG-X_6-8-QEE-catalytic motif shared by PARG in the Macro Domain family.（2）In order to find the possible catalytic site of MavL and its enzymatic cleavage mechanism,we prepared crystals of the MavL stable truncator MavL（40-404）protein in complex with ADPr molecule and resolved the structure of the complex with a resolution of 2.35（?）.The structural comparison revealed that four amino acids,D315,N322,D323,and D333,have an important role in the substrate cleavage process.The four amino acids D315,N322,D323 and D333 have important roles in the substrate cleavage process.Therefore,we designed a series of mutants based on the structure and performed in vitro digestion experiments and ITC experiments using ADPr-Ub as its substrate protein.The results showed that the mutation of all four sites reduced the enzymatic cleavage activity of MavL in vitro,and compared with the ITC results,we found that D315A had relatively strong ADPr-Ub binding ability and relatively weak ADPr-Ub hydrolysis ability.（3）Then we further prepared the crystals of the MavL_D315A-ADPr-Ub complex and resolved the structure of the complex with a resolution of 1.90（?）.Structural analysis revealed that the D323 site may play a major catalytic role in the catalytic process;D315,N322,and D333 play a role in stabilizing the distal ribosyl group in ADPr within the catalytic pocket,and K236 plays a role in outside the catalytic pocket interacts with the ADPr furanose,while H142 and Y265 sandwich the adenine in ADPr to formπ-πinteractions to stabilize the adenosine portion of ADPr.At the same time,we found that MavL can perform normal enzymatic functions only if the distal ribosyl group of ADPr is stably anchored in the catalytic pocket.（4）While clarifying that MavL has ADP ribosyl hydrolase activity,we also explored the possible substrate proteins of MavL in the host cell.We have explored the interaction of SDHA,PRDX1 and ANXA2 proteins with MavL using immunoprecipitation and other methods,unfortunately we have not found a definitive MavL substrate protein yet.We are continuing our experiments on this part to complement the mechanism of host regulation by Legionella pneumophila using ADP ribosylation modifications.