Members of the Legionella pneumophila Sde family target tyrosine residues for phosphoribosyl-linked ubiquitination.

Legionella pneumophila establishes a replication vacuole by translocating hundreds of protein effectors through a type IV secretion system (T4SS). Among these translocated effectors are members of the Sde family, which catalyze phosphoribosyl-linked ubiquitination (pR-Ub) of host targets. Previous work has posited that Sde proteins solely target serine (Ser) residues within acceptor protein substrates. We show here that SdeC-mediated pR-Ub modification results from a stepwise reaction that also modifies tyrosine (Tyr) residues. Unexpectedly, the presence of an HA tag on Ub resulted in poly-pR-ubiquitination, consistent with the HA tag acting as an acceptor target. Interrogation of phosphoribosyl-linked HA-Ub revealed that Tyr4 was the preferred targeted residue, based on LC-MS/MS analysis of the crosslinked product. Further analysis using synthetic HA variants revealed promiscuous modification of Tyr, as crosslinking was prevented only by constructing a triple mutant in which all three Tyr within the HA sequence were substituted with Phe. Although previous work has indicated that Ser is the sole acceptor residue, we found no evidence of Ser preference over Tyr using Tyr → Ser replacement mutants. This work demonstrates that pR-ubiquitination by the Sde family is not limited to Ser-modification as previously proposed, and broadens the potential sites targeted by this family.