Ubiquitin transfer by a RING E3 ligase occurs from a closed E2~ubiquitin conformation
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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.
Branigan , E , Penedo , C & Hay , R T 2020 , ' Ubiquitin transfer by a RING E3 ligase occurs from a closed E2~ubiquitin conformation ' , Nature Communications , vol. 11 , 2846 . https://doi.org/10.1038/s41467-020-16666-y
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