Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site
Bibliographic record
Abstract
Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of Ub regulation are conjugation through E1-E2-E3 enzymatic cascades and recognition by Ub-binding domains. An emerging theme in the Ub field is that these 2 properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to Ub's C terminus for Ub transfer reactions, conjugation enzymes often bind noncovalently and weakly to Ub at "exosites." However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected Ub-binding sites. Using a panel of Ub variants (UbVs), we identify a protein-based inhibitor that blocks Ub ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo-electron microscopy (cryo-EM) structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains. The results provide a tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic Ub-binding sites within large multiprotein complexes.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".