Identification And Mechanism Of A Bacterial Effector Deubiquitinase Specific For Linear Ub Chains

Linear ubiquitin chains are assembled by LUBAC complex and characterized by the head-to-tail linkage of ubiquitin via the C-terminal carboxyl group of the incoming ubiquitin and the N-terminal methionine of the preceding ubiquitin.Linear ubiquitin chains involved in many cellular processes,including NF-?B signaling and autophagyPathogenic bacteria and their host are locked in a constant battle.In order to proliferate in host cells,pathogenic bacteria have employed a variety of sophisticated strategies against host defense.Pathogenic bacteria have been reported to secrete effectors that harbour deubiquitinase activity into host cells to hydrolyze cellular linear ubiquitin chains and disrupt host ubiquitination signaling.All previously identified effector deubiquitinases hydrolyzed isopeptide-linked polyubiquitin chains.It has been a long-standing question whether bacterial pathogens have secreted an effector deubiquitinase to directly hydrolyze linear ubiquitin chainsIn this study,we established a screening assay to identify bacterial effector deubiquitinase and found that Legionella pneumophila effector RavD is a linear ubiquitin chain-specific deubiquitinaseTo uncover the enzymatic mechanism of RavD,we carried out structural studies.We successfully determined the crystal structure of RavD1c(1-200)-RavD orthologue from L pneumophila strain Corby-with linear diUb.Structural studies revealed that RavD is a papain-like deubiquitinase with a Cys-His-Ser catalytic triad.Two Ub-binding surface and Ub-interacting residues in RavD determined its specificity for linear ubiquitin chains.Our results showed that RavD is a unique linear ubiquitin chain-specific effector deubiquitinase.RavD was anchored to LCV by binding to LCV components PI(3)P and PI(4)P through its C-terminal and prevented linear ubiquitin chains accumulation on LCVs through its N-terminal deubiquitinase domain,which ultimately inhibited the activation of NF-?B pathway during infection.This study represented a new mechanism to inhibit the host NF-?B immune response during L.pneumophila infection.In addition,the screening assay also provided a strategy to identify more bacterial deubiquitinases.The specific deubiquitinase activity of RavD can be exploited as a useful tool to dissect linear ubiquitin chains-mediated signaling and substrates.