Structural Divergence without Functional Impact: Comparative Characterization of SARS-CoV-2 3CL-Mpro Variants Using Cleavage Site Substrates
Bibliographic record
Abstract
Abstract The SARS-CoV-2 main protease (3CLpro) is essential for viral replication and a leading antiviral target. Circulating variants accumulate substitutions on this enzyme, distant from the catalytic site. Surprisingly, mutant enzymes retain full proteolytic activity, though preserved overall activity does not exclude subtler effects on substrate recognition or selectivity, arising from distal structural perturbations. In this study, we compared the steady-state kinetics of wild-type (Wuhan) 3CLpro with enzymes from the Beta (K90R), Lambda (G15S), and Omicron (P132H) variants, using two peptide substrates representing distinct viral polyprotein cleavage sites. All four proteases displayed comparable catalytic efficiencies, similar pH-rate profiles, suggesting conservation of the catalytic mechanism despite sequence variation. The crystal structure of Omicron 3CLpro bound to an Nsp8-Nsp9 peptide revealed a conserved fold and active-site geometry, with the P132H side chain adopting a substrate-dependent conformation that rebuilt its local contacts – indicating towards how a distal substitution is accommodated without perturbing catalysis. Thermal stability measurements identified the sole distinguishing effect of P132H, with Omicron showing altered stability at elevated temperature. A screen of 31 tanshinones against 3CLpro identified T06 with K i values of 5 µM, as 3CL pro inhibitor. Thus, 3CLpro may tolerate distal substitutions through local structural adaptation, supporting its durability as an antiviral target.
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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".