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

Nature Communications
Gwen R BuelKylie J Walters

Abstract

Regulated proteolysis by proteasomes involves ~800 enzymes for substrate modification with ubiquitin, including ~600 E3 ligases. We report here that E6AP/UBE3A is distinguished from other E3 ligases by having a 12 nM binding site at the proteasome contributed by substrate receptor hRpn10/PSMD4/S5a. Intrinsically disordered by itself, and previously uncharacterized, the E6AP-binding domain in hRpn10 locks into a well-defined helical structure to form an intermolecular 4-helix bundle with the E6AP AZUL, which is unique to this E3. We thus name the hRpn10 AZUL-binding domain RAZUL. We further find in human cells that loss of RAZUL by CRISPR-based gene editing leads to loss of E6AP at proteasomes. Moreover, proteasome-associated ubiquitin is reduced following E6AP knockdown or displacement from proteasomes, suggesting that E6AP ubiquitinates substrates at or for the proteasome. Altogether, our findings indicate E6AP to be a privileged E3 for the proteasome, with a dedicated, high affinity binding site contributed by hRpn10.

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Citations

Jul 8, 2020·Molecular and Cellular Biology·Vasty Osei-AmponsaKylie J Walters
Nov 20, 2020·The FEBS Journal·Xiang ChenKylie J Walters
Feb 20, 2021·Pharmacology & Therapeutics·Sunil SinghJ Sivaraman

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Methods Mentioned

BETA
ubiquitination
NMR
myc-immunoprecipitation
isothermal titration calorimetry
immunoprecipitation
co-immunoprecipitation
co-immunoprecipation
transfection
circular dichroism
deubiquitination

Key Resources (RRID) Mentioned

Addgene_48138

Software Mentioned

CONTIN
DichroWeb
RAZUL
MOLMOL
XEASY
Linux
PyMOL Molecular Graphics System
Biacore T200
- NMR
Proteome Discoverer

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