Inhibition of bacterial ubiquitin ligases by SidJ/Calmodulin-catalyzed glutamylation

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.