Photocrosslinking Activity-Based Probes to Capture the Dynamics of Ubiquitin RING E3 Ligase Interactions

Abstract

Almost all cellular processes are influenced by ubiquitination. A large family of enzymes known as E3 ligases provide the specificity for ubiquitination, with the largest class among them, the RING E3s, comprising over 600 members in humans. RING E3s facilitate transfer of ubiquitin to substrates by constraining the highly dynamic E2-Ub thioester linkage to be primed for attack from the substrate nucleophile. We have established a workflow that uses a modified ubiquitin carrying a photoactivatable crosslinker that once stably linked to the active site of an E2, creates an activity based probe (ABP) to monitor interactions with E3 ligases. Using this, regions of interaction between ubiquitin and a selection of different RING E3 were determined, which not only confirmed existing structures of E2-Ub-RING-E3 complexes but was also used to assess new Ub-E2-E3 models generated in absence of existing structures.