Abstract
The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away. This activity is dependent on formation of self-anchored, K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. Here we reveal how TRIM21 facilitates ubiquitin transfer and differentiates this E2 from other closely related enzymes. A tri-ionic motif provides optimally distributed anchor points that allow TRIM21 to wrap an Ube2N~Ub complex around its RING domain, locking the closed conformation and promoting ubiquitin discharge. Mutation of these anchor points inhibits ubiquitination with Ube2N/Ube2V2, viral neutralization and immune signalling. We show that the same mechanism is employed by the anti-HIV restriction factor TRIM5 and identify spatially conserved ionic anchor points in other Ube2N-recruiting RING E3s. The tri-ionic motif is exclusively required for Ube2N but not Ube2D1 activity and provides a generic E2-specific catalysis mechanism for RING E3s.
Highlights
The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away
Antibody-dependent virus neutralization by TRIM21 is dependent on interaction with its canonical E2 enzymes Ube2W and Ube2N(Ubc13)/Ube2V2(Uev2)[2,5]
It has been suggested that an additional E2 might be required in order to build K48-chains, which are classically associated with proteasomal recruitment[2]
Summary
The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away This activity is dependent on formation of self-anchored, K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. In addition to its unusual properties of indirect substrate engagement and self-ubiquitination-driven target degradation, TRIM21 does not conform to many of the general rules of E2:E3s given above It retains activity as a monomeric RING and uses a domain uniquely found in TRIMs, the B-box, to prevent constitutive ubiquitination by blocking E2 recruitment[13]. X-ray crystallography and NMR spectroscopy reveal a tri-ionic motif that captures E2~Ub complexes in the active closed state by providing conformation-specific anchor points We show that this tri-ionic motif is conserved across TRIM RING ligases, and is used by the antiretroviral TRIM5 to drive K63-ubiquitin chain synthesis. We identify structurally conserved charged anchor points across divergent RINGs, suggesting that this may be a common mechanism for Ube2N-specific catalysis
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