E6AP AZUL interaction with UBQLN1/2 in cells, condensates, and an AlphaFold-NMR integrated structure.

The E3 ligase E6AP/UBE3A has a dedicated binding site in the 26S proteasome provided by the RAZUL domain of substrate receptor hRpn10/S5a/PSMD4. Guided by RAZUL sequence similarity, we test and demonstrate here that the E6AP AZUL binds transiently to the UBA of proteasomal shuttle factor UBQLN1/2. Despite a weak binding affinity, E6AP AZUL is recruited to UBQLN2 biomolecular condensates in vitro and E6AP interacts with UBQLN1/2 in cellulo. Steady-state and transfer nuclear Overhauser effect (NOE) experiments indicate direct interaction of AZUL with UBQLN1 UBA. Intermolecular contacts identified by NOE spectroscopy (NOESY) data were combined with AlphaFold2-Multimer predictions to yield an AZUL:UBA model structure. We additionally identify an oligomerization domain directly adjacent to UBQLN1/2 UBA (UBA adjacent [UBAA]) that is α-helical and allosterically reconfigured by AZUL binding to UBA. These data lead to a model of E6AP recruitment to UBQLN1/2 by AZUL:UBA interaction and provide fundamental information on binding requirements for interactions in condensates and cells.