Structure of the human UBR5 E3 ubiquitin ligase

Abstract

The human UBR5 (also known as EDD) is a single polypeptide chain HECT-type E3 ubiquitin ligase essential for embryonic development in mammals. Although widely expressed, UBR5 is markedly amplified and overexpressed in breast, ovarian, prostate, gastric and pancreatic cancers. Dysregulated UBR5 functions like an oncoprotein to promote cancer growth and metastasis, making UBR5 a potential target for therapeutics. Unexpectedly, we found that human UBR5 assembles a dimer and a tetramer in solution. We determined the dimer structure at 2.8 Å and the tetramer structure at 3.5 Å average resolution. UBR5 is a crescent shaped molecule with a seven-bladed β-propeller and two small β-barrel domains (SBB1/2) at the N-terminal region, a catalytic HECT domain at the C-terminus, and an extended helical scaffold and an N-degron-recognizing UBR box in the middle. The dimer is assembled as a stable head-to-tail dimer via extensive interactions in the middle helical scaffold region. The tetramer is assembled via SBB2-SBB2 interaction from two face-to-face dimers, forming a large cage with all four catalytic HECT domains facing the central cavity. Importantly, the N-terminal region of one subunit and the HECT of the other form an intermolecular jaw in the dimer. Using enzymatic and cellular assays, we showed that the jaw-lining residues are important for function, suggesting that the intermolar jaw functions to recruit ubiquitin loaded E2 to UBR5 for the transthiolation reaction. Further work is needed to understand how oligomerization regulates the UBR5 ligase activity. This work provides a framework for structure-based anticancer drug development against the distinctive HECT E3 ligase and contributes to a growing appreciation of E3 ligase diversity.

Cryo‐EM structure of the chain‐elongating E3 ubiquitin ligase UBR5

“…The elegant assembly of UBR5 protomers into dimers and tetramers may function in positioning each protein-binding module in the vicinity of a catalytic HECT domain rather than directly mediating processivity. The ring-shaped architecture is reminiscent of several monomeric and multimeric E3 enzymes such as HUWE1, GID, BIRC6 or the SCF, where this shape promotes substrate recruitment and ubiquitination (Grabarczyk et Furthermore, our interpretation of the UBR5 structure agrees well with the findings of the recently published study, which similarly portrays UBR5 as a homodimer that further assembles into a tetramer formation (Wang et al, 2023). There is an agreeable consistency between these structures, aside from the extent of the dimerisation interface.…”

The G1/S transition is promoted by Rb degradation via the E3 ligase UBR5

“…UBR5 likely engages its substrates as a dimer or tetramer, which can target distinct degron linear motifs as indicated by recent cryo-EM structures 52,53,[59][60][61] . Since UBR5 is a large multi-domain protein, it is possible that different docking mechanisms can be utilized for engaging different groups of substrates 51,53,[60][61][62] . Interestingly, two recent studies proposed that UBR5 targets its substrates on chromatin 52,53 .…”

Targeting UBR5 inhibits postsurgical breast cancer lung metastases by inducing CDC73 and p53 mediated apoptosis

“…Protein UBR5 and UBR5 C2768A expression and purification were conducted as previously described 15 . Proteins were purified using a Superdex 200 column.…”

“…Protein UBR5 and UBR5 C2768A expression and purification were conducted as previously described. 15 Proteins were purified using a Superdex 200 column. The N-terminal 6xHis-tag on Ub was cleaved and purified using a Superdex 75 column (GE Healthcare).…”