Abstract
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.
Highlights
The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamylε-Lys (Gln40Ub-Lys92Ube2N) isopeptide crosslink using a transglutaminase mechanism
Though an approximate Kd of ~170 μM for binding of free Ub to MavC was deduced from biolayer interferometry (BLI) measurements, reaction assays and NMR experiments suggest the affinity is much weaker: reaction assays monitoring Ub deamidation as a function of Ub concentration failed to saturate activity even at 375 μM Ub, suggesting that the KM for free Ub in this reaction is in excess of 200 μM (Fig. 2b, Supplementary Fig. 2a, b)
Reactions using free Ub and Ube2N at the same subunit concentrations as the disulfide conjugate (25 μM) produced no detectable transglutaminase product (Fig. 2e). These results strongly argue that MavC targets the Ube2N–Ub conjugate to catalyze an intramolecular transglutaminase reaction resulting in the formation of an isopeptide bond between Ube2N and Ub
Summary
The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamylε-Lys (Gln40Ub-Lys92Ube2N) isopeptide crosslink using a transglutaminase mechanism. Despite lacking a Ub system of their own, many pathogenic bacteria have evolved enzymes or substrate adaptors with the ability to interact with the Ub-signaling system of their eukaryotic hosts, allowing them to take control of host processes and modulate them for their benefit[5,6] Injected into their host cytoplasm through specialized secretion systems, various bacterial effectors have been found to use an array of strategies to hijack or exploit Ub-signaling pathways. In recent years, our understanding of this post-translational modification has been redefined by the discovery of bacterial enzymes that catalyze Ub transfer using strategies that bypass the canonical E1–E2–E3 pathway This was first demonstrated for the SidE family of Legionella effectors that catalyze NAD+-assisted phospho-ribosyl linked ubiquitination of certain host targets[14,15,16,17]. No clear demonstration of activity on this specific substrate was provided
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