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.
The 26S proteasome is a highly complex 2.5 MDa molecular machine responsible for regulated protein degradation. Proteasome substrates are typically marked by ubiquitination for recognition at receptor sites contributed by Rpn1/S2/PSMD2, Rpn10/S5a, and Rpn13/Adrm1. Each receptor site can bind substrates directly by engaging conjugated ubiquitin chains or indirectly by binding to shuttle factors Rad23/HR23, Dsk2/PLIC/UBQLN, or Ddi1, which contain a ubiquitin-like domain (UBL) that adopts the ubiquitin fold. Previous structural studies have defined how each of the proteasome receptor sites bind to ubiquitin chains as well as some of the interactions that occur with the shuttle factors. Here, we define how hRpn10 binds to the UBQLN2 UBL domain, solving the structure of this complex by NMR, and determine affinities for each UIM region by a titration experiment. UBQLN2 UBL exhibits 25-fold stronger affinity for the N-terminal UIM-1 over UIM-2 of hRpn10. Moreover, we discover that UBQLN2 UBL is fine-tuned for the hRpn10 UIM-1 site over the UIM-2 site by taking advantage of the additional contacts made available through the longer UIM-1 helix. We also test hRpn10 versatility for the various ubiquitin chains to find less specificity for any particular linkage type compared to hRpn1 and hRpn13, as expected from the flexible linker region that connects the two UIMs; nonetheless, hRpn10 does exhibit some preference for K48 and K11 linkages. Altogether, these results provide new insights into the highly complex and complementary roles of the proteasome receptor sites and shuttle factors.
Highlights d Chemical origin of Rpn13 preference for K48-linked ubiquitin chains revealed d NMR demonstrates highly dynamic interactions between hRpn2:hRpn13 and K48-diubiquitin d K48-diubiquitin adopts a dynamic, extended conformation that hRpn13 selects d Structure of hRpn2:Rpn13 bound to K48-diubiquitin described
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...
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