Abstract: The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein
modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens
exploit this post-translational modification process by encoding molecular mimics of
E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade.
Here, we show that the Legionella pneumophila effector protein RavN belongs to a
growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation
pathway. The E3 ligase activity of RavN was located within its N-terminal region and was
dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The
crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only
remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has
undergone significant evolutionary alteration. Substitution of residues within the predicted
E2 binding interface rendered RavN inactive, indicating that, despite significant structural
changes, the mode of E2 recognition has remained conserved. Using hidden Markov
model-based secondary structure analyses, we identified and experimentally validated four
additional L. pneumophila effectors that were not previously recognized to possess E3
ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong
evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to
the manipulation of the host ubiquitylation pathway.