Mechanism of Lys6 poly-ubiquitin specificity by the L. pneumophila deubiquitinase LotA

The versatility of ubiquitination to control vast domains of eukaryotic biology is due, in part, to diversification through differently linked poly-ubiquitin chains. Deciphering signaling roles for some chain types, including those linked via K6, has been stymied by a lack of specificity among the implicated regulatory proteins. Forged through strong evolutionary pressures, pathogenic bacteria have evolved intricate mechanisms to regulate host ubiquitin during infection. Herein, we identify and characterize a deubiquitinase domain of the secreted effector LotA from Legionella pneumophila that specifically regulates K6-linked poly-ubiquitin. We demonstrate the utility of LotA for studying K6 poly-ubiquitin signals. We identify the structural basis of LotA activation and poly-ubiquitin specificity and describe an essential “adaptive” ubiquitin-binding domain. Without LotA activity during infection, the Legionella-containing vacuole becomes decorated with K6 poly-ubiquitin as well as the AAA ATPase VCP/p97/Cdc48. We propose that LotA’s deubiquitinase activity guards Legionella-containing vacuole components from ubiquitin-dependent extraction. [Display omitted] •L. pneumophila LotA encodes two functionally distinct deubiquitinases•LotA’s specificity for K6-linked poly-ubiquitin makes it a valuable research tool•Structures reveal that K6 specificity arises from substrate-assisted catalysis•LotA restricts K6 poly-ubiquitin and VCP recruitment during infection Warren et al. identify a K6-specific deubiquitinase within the Legionella pneumophila effector LotA and demonstrate its utility as a research tool. Structural and biochemical work explain the mechanism of LotA activation and specificity. During infection, LotA’s deubiquitinase activity guards the Legionella-containing vacuole from K6 poly-ubiquitin and VCP recruitment.