RT Journal Article SR Electronic T1 Novel class of OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.04.25.060954 DO 10.1101/2020.04.25.060954 A1 Donghyuk Shin A1 Anshu Bhattacharya A1 Yi-Lin Cheng A1 Marta Campos Alonso A1 Ahmad Reza Mehdipour A1 Gerbrand J. van der Heden van Noort A1 Huib Ovaa A1 Gerhard Hummer A1 Ivan Dikic YR 2020 UL http://biorxiv.org/content/early/2020/04/28/2020.04.25.060954.abstract AB Legionella pneumophila is a gram-negative pathogenic bacterium that causes Legionaries’ disease. The Legionella genome codes more than 300 effector proteins able to modulate host-pathogen interactions during infection. Among them are also enzymes altering the host-ubiquitination system including bacterial ligases and deubiquitinases. In this study, based on homology-detection screening on 305 Legionella effector proteins, we identified two Legionella OTU-like deubiquitinases (LOT; LotB (Lpg1621/Ceg23) and LotC (Lpg2529), LotA (Lpg2248/Lem21) is already known). A crystal structure of LotC catalytic core (LotC14-310) was determined at 2.4 Å and compared with other OTU deubiquitinases, including LotB. Unlike the classical OTU-family, the structures of Legionella OTU-family (LotB and LotC) shows an extended helical lobe between the Cys-loop and the variable loop, which define a novel class of OTU-deubiquitinase. Despite structural differences in their helical lobes, both LotB and LotC interact with ubiquitin. LotB has an additional ubiquitin binding site (S1’) enabling specific cleavage of Lys63-linked poly-ubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of catalytically inactive LotB and LotC identified different categories of host-substrates for these two related DUBs. Together, our results provide new structural insights of bacterial OTU deubiquitinases and indicate distinct roles of bacterial deubiquitinases in host-pathogen interactions.Competing Interest StatementThe authors have declared no competing interest.