Crystal structure and activity-based labeling reveal the mechanisms for linkage-specific substrate recognition by deubiquitinase USP9X
Bibliographic record
Abstract
USP9X is a conserved deubiquitinase (DUB) that regulates multiple cellular processes. Dysregulation of USP9X has been linked to cancers and X-linked intellectual disability. Here, we report the crystal structure of the USP9X catalytic domain at 2.5-Å resolution. The structure reveals a canonical USP-fold comprised of fingers, palm, and thumb subdomains, as well as an unusual β-hairpin insertion. The catalytic triad of USP9X is aligned in an active configuration. USP9X is exclusively active against ubiquitin (Ub) but not Ub-like modifiers. Cleavage assays with di-, tri-, and tetraUb chains show that the USP9X catalytic domain has a clear preference for K11-, followed by K63-, K48-, and K6-linked polyUb chains. Using a set of activity-based diUb and triUb probes (ABPs), we demonstrate that the USP9X catalytic domain has an exo-cleavage preference for K48- and endo-cleavage preference for K11-linked polyUb chains. The structure model and biochemical data suggest that the USP9X catalytic domain harbors three Ub binding sites, and a zinc finger in the fingers subdomain and the β-hairpin insertion both play important roles in polyUb chain processing and linkage specificity. Furthermore, unexpected labeling of a secondary, noncatalytic cysteine located on a blocking loop adjacent to the catalytic site by K11-diUb ABP implicates a previously unreported mechanism of polyUb chain recognition. The structural features of USP9X revealed in our study are critical for understanding its DUB activity. The new Ub-based ABPs form a set of valuable tools to understand polyUb chain processing by the cysteine protease class of DUBs.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 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) | 0.000 | 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 teacher head, 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".