Noort, G. J. v. d. H. v. | Search bioRxiv

bioRxiv2023

Structural insights into ubiquitin chain cleavage by Legionella ovarian tumor deubiquitinases

Although ubiquitin is found only in eukaryotes, several pathogenic bacteria and viruses possess proteins that hinder the host ubiquitin system. Legionella, a gram-negative intracellular bacterium, possesses an ovarian tumor (OTU) family of deubiquitinases (Lot DUBs). Herein, we describe the molecular characteristics of Lot DUBs. We elucidated the structure of LotA OTU1 domain and revealed that entire Lot DUBs possess a characteristic extended helical lobe (EHL) that is not found in other OTU-DUBs. The structural topology of EHL is the same throughout the Lot family, and it provides an S1' ubiquitin-binding site. Moreover, the catalytic triads of Lot DUBs resemble those of the A20-type OTU-DUBs. Furthermore, we revealed a unique mechanism by which LotA OTU domains cooperate together to distinguish the length of the chain and preferentially cleaves longer K48-linked polyubiquitin chains. The LotA OTU1 domain itself cleaves K6-linked ubiquitin chains, while it is also essential for assisting the cleavage of longer K48-linked polyubiquitin chains by the OTU2 domain. Thus, this study provides novel insights into the structure and mechanism of action of Lot DUBs.

biochemistry↗

bioRxiv2020

Novel class of OTU deubiquitinases regulate substrate ubiquitinationupon Legionella infection

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 [A] 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.

molecular biology↗