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Highlights d Phosphoribosyl serine ubiquitination can be reverted by DupA and DupB d DUP specifically binds to and cleaves PR-ubiquitin from serine on substrates d Catalytically inactive DupA mutants can capture PRubiquitinated proteins d PR ubiquitination on multiple ER structural proteins causes ER fragmentation

The family of bacterial SidE enzymes catalyzes phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophilia , a pathogenic bacterium causing Legionnaires’ disease 1 , 2 , 3 . SidEs share the genetic locus with the Legionella effector SidJ that spatiotemporally opposes their toxicity in yeast and mammalian cells, through an unknown mechanism 4 – 6 . Deletion of SidJ leads to a significant defect in the growth of Legionella in both its natural host amoeba and in murine macrophages 4 , 5 . Here, we demonstrate that SidJ is a glutamylase that modifies the catalytic glutamate in the mono-ADPribosyl transferase (mART) domain of SdeA thus blocking its ubiquitin (Ub) ligase activity. SidJ glutamylation activity requires interaction with Calmodulin (CaM), a eukaryotic specific co-factor, and can be regulated by intracellular changes in Ca 2+ concentrations. The cryo-EM structure of SidJ/human apo-CaM complex revealed the architecture of this unique heterodimeric glutamylase. In infected cells, we show that SidJ mediates glutamylation of SidEs on the surface of Legionella- containing vacuoles (LCVs). Using quantitative proteomics, we also uncovered multiple host proteins as putative targets of SidJ-mediated glutamylation. Collectively, this study reveals the mechanism of SidE ligases inhibition by a SidJ/CaM glutamylase and opens new avenues for studying protein glutamylation, an understudied protein modification in higher eukaryotes.

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.