Abstract
Legionella pneumophila causes a severe pneumonia known as Legionnaires' disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two LegionellaOTU (ovarian tumor)-like deubiquitinases (LOT-DUBs; LotB [Lpg1621/Ceg23] and LotC [Lpg2529]). The crystal structure of the LotC catalytic core (LotC14-310) was determined at 2.4 Å. Unlike the classical OTU-family, the LOT-family shows an extended helical lobe between the Cys-loop and the variable loop, which defines them as a unique class of OTU-DUBs. LotB has an additional ubiquitin-binding site (S1'), which enables the specific cleavage of Lys63-linked polyubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of LotB and LotC identified different categories of host-interacting proteins and substrates. Together, our results provide new structural insights into bacterial OTU-DUBs and indicate distinct roles in host-pathogen interactions.
Highlights
Ubiquitination, a well-studied post-translational modification, regulates various cellular events (Yau and Rape, 2016)
Using pairwise sequence-structure comparison based on hidden Markov models (HMMs, HHpred suite) (Zimmermann et al, 2018), we revealed four previously uncharacterized proteins as putative DUBs
LotB only partially shifted after 30 min, as evident by the amount of unreacted species, while LotC rapidly reacted with Prg-activity-based probes (ABPs) and was completely conjugated after 30 min (Figure 2d and e). These results suggest that both LotB and LotC have a primary ubiquitin-binding S1 site, where the propargyl group can be located in close proximity to the catalytic cysteine
Summary
Ubiquitination, a well-studied post-translational modification, regulates various cellular events (Yau and Rape, 2016). A representative example of the ubiquitin-mediated cellular process is the ubiquitin-proteasome system, where misfolded proteins get ubiquitinated, degraded by the proteasome, and, recycled (Dikic, 2017). To maintain homeostasis in the cell, ubiquitination events are tightly regulated by a reverse process called deubiquitination, where ubiquitin molecules are cleaved from the target substrates and subsequently recycled by deubiquitinating enzymes (deubiquitinases [DUBs]) (Clague et al, 2019). About 100 different DUBs have been identified in human They are categorized into seven different classes based on their structure and mechanism of action, and these include USP, JAMM (MPN), OTU, MJD (Josepin), UCH, and the recently discovered MINDY and ZUFSP
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