Photocross-linking activity-based probes to capture the dynamics of ubiquitin RING E3 ligase interactions

Almost all cellular processes are influenced by ubiquitination. A large family of enzymes known as E3 ligases provides the specificity for ubiquitination, with the largest class among them, the Really Interesting New Gene (RING) E3s, comprising over 600 members in humans. RING E3s facilitate transfer of ubiquitin (Ub) 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 an N-maleimido diazirine (NMD) photoactivatable cross-linker attached to ubiquitin that, once stably linked to the active site of an E2, creates an activity-based probe (ABP) to monitor interactions with E3 ligases. Cross-linking mass spectrometry using the NMD-Ub-E2 ABP identified regions of interaction between ubiquitin and a selection of different RING E3s, which not only agreed with existing crystal structures, but was also used to evaluate in silico structural models of complexes yet to be resolved by conventional means. The cross-linking data also provided insight into domains of conformational flexibility which likely adopt multiple configurations in solution and which are challenging to monitor by other methods. NMD-Ub-E2 ABPs offer great potential to explore the ensemble conformations of E2-E3 complexes in solution and have scope for applications beyond the ubiquitin system.