Structure
ArticleE6AP AZUL interaction with UBQLN1/2 in cells, condensates, and an AlphaFold-NMR integrated structure
Graphical abstract
Introduction
The ubiquitin-proteasome pathway removes proteins that are misfolded or no longer needed in cells.1 Its substrates are marked for degradation by post-translational modification with ubiquitin.2 Ubiquitination begins with ATP-dependent charging of the ubiquitin C terminus by an E1 activating enzyme for subsequent thioester transfer to an E2 conjugating enzyme. An E3 ligase next acts as either a scaffold to facilitate direct transfer of ubiquitin from the E2 to a substrate or as an intermediary receptor by first accepting ubiquitin from the E2 before passing it to the substrate. The E3 ligase E6AP/UBE3A belongs to the latter class of E3s and is the namesake of this protein family called homologous to the E6AP carboxyl terminus (HECT) E3s. E6AP is infamous for its roles in human disease; human papilloma viral (HPV) oncoprotein E6 binds E6AP and directs its activity toward tumor suppressor p53, contributing to cervical cancer.3,4,5 E6AP can also promote metastatic prostate cancer6,7 and is implicated in neurological disorders, with loss-of-function mutations linked to Angelman syndrome8,9,10 and elevated gene dosage with autism spectrum disorders.11
An amino-terminal zinc-binding domain of ubiquitin E3a ligase (AZUL) domain12 in E6AP binds to an intrinsically disordered region in the proteasome ubiquitin receptor protein hRpn10/S5a/PSMD4, named Rpn10 AZUL-binding domain (RAZUL).13 Binding to E6AP AZUL causes RAZUL to form two α helices that interact with two AZUL α helices to form a four-helix bundle, and loss of this interaction leads to loss of proteasome-associated E6AP.13 E6AP has three isoforms with distinct localization to the nucleus or cytosol in neurons.14,15 Nuclear E6AP localization is contingent on AZUL-mediated interaction with hRpn10, and E6AP mislocalization causes physiological defects.15 Other E3 ligases associate with the proteasome13,16,17,18,19 but without known binding mechanisms.
Nuclear E6AP and proteasomes co-localize to biomolecular condensates that are induced by hyperosmotic stress or nutrient deprivation and require RAD23B and ubiquitinated proteins.20,21 RAD23B and closely related Rad23A belong to a larger family of shuttle factor proteins, so named by their ability to deliver ubiquitinated proteins to the proteasome, that also includes DDI1/2 and UBQLN proteins (UBQLN1-4 and UBQLNL).22 RAD23B and UBQLN1/2 are found associated with proteasomes purified from cells18,23,24 and can stimulate proteasomal ATP hydrolysis and proteolysis25 through a mechanism that has not yet been elucidated. UBQLN proteins can recruit an E3 ligase of unknown identity to ubiquitinate bound substrates through an interaction involving the ubiquitin-associated (UBA) domain,26 which is known to bind ubiquitin27,28,29 and contribute to interaction with the proteasome.30
The UBA in UBQLN2 contributes to the ability of UBQLN2 to form biomolecular condensates,31,32 and RAD23B’s two UBA domains similarly drive formation of nuclear condensates containing proteasomes.20 K48-linked ubiquitin chains appear to drive formation of RAD23B condensates,20 and K48- or K63-linked ubiquitin chains slightly or strongly promote UBQLN2 condensate formation, respectively.33
Here, we find that a C-terminal region of UBQLN1/2 that includes its UBA domain has sequence similarity to the hRpn10 RAZUL, with conservation of amino acids involved in binding to E6AP. We use nuclear magnetic resonance (NMR) spectroscopy to test and confirm that the E6AP AZUL binds to the UBQLN1 UBA region. We find evidence of this interaction in cells and observe association of the E6AP AZUL with UBQLN2 condensates in an in vitro assay. By integrating NMR and biophysical data with AlphaFold2-Multimer, we generate a structural model of the UBA:AZUL complex and of a UBA-adjacent (UBAA) domain that is helical and self-associates. Together, our data suggest that the E6AP AZUL binds to the UBQLN1/2 UBA and this interaction allosterically affects UBQLN UBAA self-association.
Section snippets
E6AP binds to UBQLN1 and UBQLN2 in cells
Following our discovery that E6AP binds to hRpn10 RAZUL through its AZUL domain,13 we searched for proteins with sequence similarity to the RAZUL domain to identify other potential binders of E6AP AZUL. This approach identified a region with 34.1% and 28.6% identity (51.2% and 50% similarity) to RAZUL within UBQLN1 and UBQLN2 respectively (Figure 1A). These two isoforms are the most closely related of the UBQLN proteins (Figure S1A), with 88% sequence identity in the identified region (Figure 1
Discussion
In this study, we discover that E6AP interacts with UBQLN1 and UBQLN2 in vitro and in cellulo and establish a structural model of their interaction by using AlphaFold2-Multimer in combination with intermolecular NOESY data. While we do not have any data investigating whether the E6AP AZUL also interacts with other UBQLNs, we expect the AZUL may also interact with UBQLN3 and UBQLN4 based on high sequence similarity in the region of interaction. We find that, although UBQLN interaction with AZUL
Key resources table
REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-E6AP (immunoblot) Sigma-Aldrich Cat# E8655; RRID: AB_261956 Rabbit polyclonal anti-E6AP (Immunoprecipitation) ProteinTech Cat# 10344-1-AP; RRID: AB_2211801 Mouse monoclonal anti-UBQLN2 (immunoblot) Novus Biologicals Cat# NBP2-25164; RRID: AB_2885154 Rabbit monoclonal anti-UBQLN2 (immunoprecipitation) Cell Signaling Technology Cat# 85509; RRID: AB_2800056 Rabbit monoclonal anti-UBQLN1 Cell Signaling Technology Cat# 14526; RRID: AB_2798502
Acknowledgments
This work was supported by the Intramural Research Program through the Center for Cancer Research, National Cancer Institute, National Institutes of Health (1ZIABC011627), and the Center for Cancer Research FLEX program (to K.J.W.), and in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract 75N91019D00024 (to H.M.). W.M. was supported in part by the NIH Office of Intramural Training and Education’s Intramural AIDS Research Fellowship, and
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