Legionella effector MavC targets the Ube2N∼Ub conjugate for noncanonical ubiquitination

The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a gamma-glutamyl-epsilon-Lys (Gln40(Ub)-Lys92(Ube2N)) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N similar to ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2 similar to Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2 similar to Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme's true physiological substrate. The Legionella pneumophila effector MavC inhibits the human ubiquitin-conjugating enzyme Ube2N. Here, the authors combine NMR, X-ray crystallography and biochemical assays and show that MavC catalyses the intramolecular transglutaminase reaction between the Ube2N and Ub subunits of the Ube2N similar to Ub conjugate and present the substrate- and product-bound MavC crystal structures.