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Discovery of E6AP AZUL binding to UBQLN1/2 in cells, phase-separated droplets, and an AlphaFold-NMR integrated structure

Buel, G. R.; Chen, X.; Myint, W.; Kayode, O.; Folimonova, V.; Cruz, A.; Skorupka, K.; Matsuo, H.; Walters, K.

2022-09-30 biophysics
10.1101/2022.09.29.510132 bioRxiv
Show abstract

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 phase-separated droplets and E6AP interacts with UBQLN1/2 in cells. Steady-state and transfer NOE experiments indicate direct interaction of AZUL with the UBQLN1 UBA domain. Intermolecular contacts identified by NOESY data were combined with AlphaFold2-Multimer predictions to yield an AZUL:UBA model structure. We also identify a concentration-dependent oligomerization domain directly adjacent to UBQLN1/2 UBA (UBA-adjacent, UBAA) that is -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 droplets and cells.

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