Regulation of phosphoribosyl ubiquitination by a calmodulin-dependent glutamylase

The bacterial pathogen Legionella pneumophila creates an intracellular niche permissive for its replication by extensively modulating host cell functions using hundreds of effector proteins delivered via its Dot/Icm secretion system1. Among these, members of the SidE family (SidEs) regulate multiple cellular processes by a unique phosphoribosyl ubiquitination mechanism that bypasses the canonical ubiquitination machinery2–4. The activity of SidEs is regulated by SidJ, another Dot/Icm effector5, but the mechanism of such regulation is not completely understood6,7. Here we demonstrate that SidJ inhibits the activity of SidEs by inducing covalent attachment of glutamate moieties to E860 of SdeA, which is one of the catalytic residues for the mono-ADP-ribosyltransferase activity involved in ubiquitin activation2. The inhibition by SidJ is spatially restricted in host cells because its activity requires the eukaryote-specific protein calmodulin (CaM). We solved a structure of SidJ-CaM in complex with adenosine monophosphate (AMP) and found that the ATP utilized is cleaved at the α phosphate position by SidJ which in the absence of glutamate or modifiable SdeA undergoes self-AMPylation. Our results reveal an unprecedented mechanism of regulation in bacterial pathogenicity in which a glutamylation reaction that inhibits the activity of virulence factors is activated by host factor-dependent acyl-adenylation.