Cocrystallization of ubiquitin-deubiquitinase complexes through disulfide linkage.

Structural characterization of the recognition of ubiquitin (Ub) by deubiquitinases (DUBs) has largely relied on covalent complexation of the DUB through its catalytic cysteine with a Ub C-terminal electrophile. The Ub electrophiles are accessed through intein chemistry in conjunction with chemical synthesis. Here, it was asked whether DUB-Ub covalent complexes could instead be accessed by simpler disulfide chemistry using a Ub cysteine mutant in which the last glycine has been replaced with a cysteine. The Ub cysteine mutant displayed a wide variability in disulfide formation across a panel of eukaryotic and prokaryotic DUBs, with some showing no detectable reaction while others robustly produced a disulfide complex. Using this approach, two disulfide-linked ubiquitin-bound complexes were crystallized, one involving the Legionella pneumophila effector SdeA DUB and the other involving the Orientia effector OtDUB. These DUBs had previously been crystallized in Ub-bound forms using the C-terminal electrophile strategy and noncovalent complexation, respectively. While the disulfide-linked SdeA DUB-Ub complex crystallized as expected, in the OtDUB complex the disulfide bond to the Ub mutant involved a cysteine that differed from the catalytic cysteine. Disulfide formation with the SdeA DUB catalytic cysteine was accompanied by local distortion of the helix carrying the active-site cysteine, whereas OtDUB reacted with the Ub mutant using a surface-exposed cysteine.

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 γ-glutamyl-ε-Lys (Gln40Ub-Lys92Ube2N) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~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~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.