Abstract
Based on extensive structural analysis it was proposed that RING E3 ligases prime the E2~ubiquitin conjugate (E2~Ub) for catalysis by locking it into a closed conformation, where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer has yet to be experimentally tested. To test this hypothesis we use single molecule Förster Resonance Energy Transfer (smFRET) to measure the conformation of a FRET labelled E2~Ub conjugate, which distinguishes between closed and alternative conformations. We describe a real-time FRET assay with a thioester linked E2~Ub conjugate to monitor single ubiquitination events and demonstrate that ubiquitin is transferred to substrate from the closed conformation. These findings are likely to be relevant to all RING E3 catalysed reactions ligating ubiquitin and other ubiquitin-like proteins (Ubls) to substrates.
Highlights
Based on extensive structural analysis it was proposed that RING E3 ligases prime the E2~ubiquitin conjugate (E2~Ub) for catalysis by locking it into a closed conformation, where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by substrate nucleophile
Crystallisation of the complex without UEV yielded the same closed conformation of the Ubc13~Ub conjugate[5] (Fig. 1b), even RNF4 fails to transfer ubiquitin from the Ubc13~Ub conjugate in the absence of UEV (Fig. 1c, d)
We found that the RNF4 RING domain dimer has a higher binding affinity for the Ubc13~Ub conjugate when in complex with UEV (Fig. 1e) suggesting that UEV plays a central role in priming of the Ubc13~Ub conjugate for catalysis when bound to the dimeric RING of RNF4
Summary
Based on extensive structural analysis it was proposed that RING E3 ligases prime the E2~ubiquitin conjugate (E2~Ub) for catalysis by locking it into a closed conformation, where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by substrate nucleophile. We describe a real-time FRET assay with a thioester linked E2~Ub conjugate to monitor single ubiquitination events and demonstrate that ubiquitin is transferred to substrate from the closed conformation. These findings are likely to be relevant to all RING E3 catalysed reactions ligating ubiquitin and other ubiquitin-like proteins (Ubls) to substrates. Using RNF4 as a RING E3 ligase, which targets SUMOylated proteins for ubiquitination, and a reactive thioester linked E2~Ub conjugate, our real-time smFRET experiment reveals that the RNF4 catalysed ubiquitination reaction of a SUMOylated substrate proceeds from the high FRET state or closed conformation of the E2~Ub conjugate
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