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

Abstract

Regulated proteolysis by the proteasome involves ~800 enzymes for substrate modification with ubiquitin, of which ~600 are E3 ligases. We report here that E6AP/UBE3A is distinguished from other ubiquitin 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, this domain in hRpn10 locks into a novel well-defined helical structure to form an intermolecular 4-helix bundle with the E6AP AZUL domain, 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 the proteasome, where associated ubiquitin is correspondingly reduced, 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. KeywordsRpn10/PSMD4/S5a; E6AP/UBE3A; proteasome; ubiquitin E3 ligase; AZUL domain 1 1 RAZUL ( Supplementary Fig. 6a), leading us to conclude that the observed difference between the theoretical and experimental CD spectra reflects increased helicity for RAZUL, consistent with the NMR data (for example, Fig. 4c). Structure of the RAZUL:AZUL complexAt the molecular interface, a 4-helix bundle is formed by two pairs of helices from AZUL and RAZUL stacking against each other (Fig. 4b). RAZUL α1 is centered between the two AZUL helices by hydrophobic interactions involving F334, L335, V338 and L339 as well as L342 and V345 from the RAZUL α1/ α2 loop ( Fig. 5a and 5b). These residues interact with A29, I33, and Y37 from AZUL H1 and A69 and L73 from AZUL H2 ( Fig. 5a and 5b). From the 3 10 -helix, V328 and M329 form hydrophobic interactions with AZUL A29, L73 and Y76 (Fig. 5c), capping the hydrophobic contact surface formed by RAZUL α1. RAZUL α2 is more peripheral compared to α1, with A351, I352, A355, M356, and L359 interacting with A67, L70 and L73 from AZUL H2 (Fig. 5d).The RAZUL N-terminal end (E322-D327) is rich in acidic residues ( Supplementary Fig. 1a) and proximal to the positively charged AZUL N-terminal end, which includes K25 and R26 (Fig. 5e).These AZUL residues contribute three hydrogen bonds to the complex, engaging RAZUL E323, D324, and Y326 (Fig. 5c). Y326, which is phosphorylated in Jurkat cells 60 , forms a hydrogen bond with the AZUL R26 sidechain in 80% of calculated structures, as well as hydrophobic contacts with AZUL K25 and R26 (Fig. 5c). We tested whether adding a bulky phosphate group at this location could be deleterious for AZUL binding by synthesizing RAZUL peptides that span E322-D366 without and with Y326 phosphorylated and measuring affinity by ITC. This shorter wild-type peptide bound with an affinity within error of hRpn10 305-377 ( Fig. 5f and Supplementary samples were validated by mass spectrometry. 26S proteasome (human) was purchased (Enzo Life Sciences, Inc. BML-PW9310). Peptide synthesishRpn10 322-3...

Impact of Losing hRpn13 Pru or UCHL5 on Proteasome Clearance of Ubiquitinated Proteins and RA190 Cytotoxicity

“…respectively (22)(23)(24)(25), whereas hRpn10 has a C-terminal domain for ubiquitin E3 ligase E6AP/UBE3A (26).…”

An Extended Conformation for K48 Ubiquitin Chains Revealed by the hRpn2:Rpn13:K48-Diubiquitin Structure

“…Substrate receptors Rpn1/S2/PSMD2, Rpn10/S5a/PSMD4, and Rpn13/Adrm1 in the RP capture ubiquitinated substrates by recognition of ubiquitin and/or shuttle factors bound to ubiquitinated substrates (Chen et al, 2016(Chen et al, , 2019Hiyama et al, 1999;Husnjak et al, 2008;Schreiner et al, 2008;Shi et al, 2016;Walters et al, 2002;Young et al, 1998;Zhang et al, 2009). These three receptors also contribute ubiquitin-processing enzymes to the proteasome, deubiquitinating enzymes Usp14 (Borodovsky et al, 2001;Leggett et al, 2002;Verma et al, 2000) and Uch37 (Lam et al, 1997) for Rpn1 and Rpn13, respectively, and ubiquitin E3 ligase E6AP for Rpn10 (Buel et al, 2020).…”