Structure
Volume 31, Issue 4, 6 April 2023, Pages 395-410.e6
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Article
E6AP AZUL interaction with UBQLN1/2 in cells, condensates, and an AlphaFold-NMR integrated structure

https://doi.org/10.1016/j.str.2023.01.012Get rights and content

Highlights

  • Discovery of E6AP AZUL interaction with UBQLN1/2 UBA domain

  • E6AP AZUL associates with UBQLN2 biomolecular condensates

  • E6AP AZUL:UBQLN1 UBA structure by merging NOE data with AlphaFold2-Multimer

  • UBA-adjacent helix self-associates and allosterically senses AZUL:UBA binding

Summary

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.

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 RESOURCESOURCEIDENTIFIER
Antibodies
Mouse monoclonal anti-E6AP (immunoblot)Sigma-AldrichCat# E8655; RRID: AB_261956
Rabbit polyclonal anti-E6AP (Immunoprecipitation)ProteinTechCat# 10344-1-AP; RRID: AB_2211801
Mouse monoclonal anti-UBQLN2 (immunoblot)Novus BiologicalsCat# NBP2-25164; RRID: AB_2885154
Rabbit monoclonal anti-UBQLN2 (immunoprecipitation)Cell Signaling TechnologyCat# 85509; RRID: AB_2800056
Rabbit monoclonal anti-UBQLN1Cell Signaling TechnologyCat# 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

References (76)

  • T.P. Dao et al.

    Ubiquitin modulates liquid-liquid phase separation of UBQLN2 via disruption of multivalent interactions

    Mol. Cell

    (2018)
  • X. Chen et al.

    Structure of hRpn10 bound to UBQLN2 UBL illustrates basis for complementarity between shuttle factors and substrates at the proteasome

    J. Mol. Biol.

    (2019)
  • S. Kühnle et al.

    Angelman syndrome-associated point mutations in the Zn(2+)-binding N-terminal (AZUL) domain of UBE3A ubiquitin ligase inhibit binding to the proteasome

    J. Biol. Chem.

    (2018)
  • K.A. Burke et al.

    Residue-by-Residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase II

    Mol. Cell

    (2015)
  • D. Zhang et al.

    Affinity makes the difference: nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains

    J. Mol. Biol.

    (2008)
  • M.F. Kleijnen et al.

    The hPLIC proteins may provide a link between the ubiquitination machinery and the proteasome

    Mol. Cell

    (2000)
  • C.D. Schwieters et al.

    The Xplor-NIH NMR molecular structure determination package

    J. Magn. Reson.

    (2003)
  • E. Ferrario et al.

    The integration of AlphaFold-predicted and crystal structures of human trans-3-hydroxy-l-proline dehydratase reveals a regulatory catalytic mechanism

    Comput. Struct. Biotechnol. J.

    (2022)
  • X. Wu et al.

    Vesicle tethering on the surface of phase-separated active zone condensates

    Mol. Cell

    (2021)
  • X. Chen et al.

    Structures of Rpn1 T1:Rad23 and hRpn13:hPLIC2 reveal distinct binding mechanisms between substrate receptors and shuttle factors of the proteasome

    Structure

    (2016)
  • J.P. Carver et al.

    General 2-site solution for chemical exchange produced dependence of T2 upon carr-purcell pulse separation

    J. Magn. Reson.

    (1972)
  • J. Jen

    Chemical exchange and Nmr T2 relaxation - multisite case

    J. Magn. Reson.

    (1978)
  • D.G. Davis et al.

    Direct measurements of the dissociation-rate constant for inhibitor-enzyme complexes via the T1 rho and T2 (CPMG) methods

    J. Magn. Reson. B

    (1994)
  • I.H. van Stokkum et al.

    Estimation of protein secondary structure and error analysis from circular dichroism spectra

    Anal. Biochem.

    (1990)
  • X. Chen et al.

    Proteasome interaction with ubiquitinated substrates: from mechanisms to therapies

    FEBS J.

    (2021)
  • J.M. Huibregtse et al.

    Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53

    Mol. Cell Biol.

    (1993)
  • J.M. Huibregtse et al.

    Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins

    Mol. Cell Biol.

    (1993)
  • P.J. Paul et al.

    Restoration of tumor suppression in prostate cancer by targeting the E3 ligase E6AP

    Oncogene

    (2016)
  • T. Kishino et al.

    UBE3A/E6-AP mutations cause Angelman syndrome

    Nat. Genet.

    (1997)
  • T. Matsuura et al.

    De novo truncating mutations in E6-AP ubiquitin-protein ligase gene (UBE3A) in Angelman syndrome

    Nat. Genet.

    (1997)
  • R.C. Samaco et al.

    Epigenetic overlap in autism-spectrum neurodevelopmental disorders: MECP2 deficiency causes reduced expression of UBE3A and GABRB3

    Hum. Mol. Genet.

    (2005)
  • A. Lemak et al.

    Zn-binding AZUL domain of human ubiquitin protein ligase Ube3A

    J. Biomol. NMR

    (2011)
  • G.R. Buel et al.

    Structure of E3 ligase E6AP with a proteasome-binding site provided by substrate receptor hRpn10

    Nat. Commun.

    (2020)
  • S. Miao et al.

    The Angelman syndrome protein Ube3a is required for polarized dendrite morphogenesis in pyramidal neurons

    J. Neurosci.

    (2013)
  • R. Avagliano Trezza et al.

    Loss of nuclear UBE3A causes electrophysiological and behavioral deficits in mice and is associated with Angelman syndrome

    Nat. Neurosci.

    (2019)
  • G. Martínez-Noël et al.

    Identification and proteomic analysis of distinct UBE3A/E6AP protein complexes

    Mol. Cell Biol.

    (2012)
  • X. Wang et al.

    Mass spectrometric characterization of the affinity-purified human 26S proteasome complex

    Biochemistry

    (2007)
  • H.C. Besche et al.

    Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates

    EMBO J.

    (2014)
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