Utilization of a Novel Recombinant Myoglobin Fusion Protein Expression System to Characterize the Tissue Inhibitor of Metalloproteinase (TIMP)-4 and TIMP-2 C-terminal Domain and Tails by Mutagenesis
Bibliographic record
Abstract
Tissue inhibitor of metalloproteinase (TIMP)-4 binds pro-matrix metalloproteinase (MMP)-2 and efficiently inhibits MT1-MMP, but unlike TIMP-2 neither forms a trimolecular complex nor supports pro-MMP-2 activation. To investigate the structural and functional differences between these two TIMPs, the C-terminal domains (C-TIMP-4 and C-TIMP-2) were expressed independently from their N domains and mutations were introduced into the C-terminal tails. Myoglobin was used as a novel recombinant fusion protein partner because spectroscopic measurement of the heme Soret absorbance at 408 nm readily enabled calculation of the molar equivalent of the red-colored recombinant protein, even in complex protein mixtures. Both C-TIMP-4 and C-TIMP-2 bound pro-MMP-2 and blocked concanavalin A-induced cellular activation of the enzyme. Measurement of k(on) rates revealed that the inhibition of MMP-2 by TIMP-4 is preceded by a C domain docking interaction, but in contrast to TIMP-2, this is not enhanced by a C-terminal tail interaction and so occurs at a slower rate. Indeed, the binding stability of C-TIMP-4 was unaltered by deletion of the C-terminal tail, but replacement with the tail of TIMP-2 increased its affinity for pro-MMP-2 by approximately 2-fold, as did substitution with the TIMP-2 C-terminal tail acidic residues in the tail of C-TIMP-4 (V193E/Q194D). Conversely, substitution of the C-terminal tail of C-TIMP-2 with that of TIMP-4 reduced pro-MMP-2 binding by approximately 75%, as did reduction of its acidic character by mutation to the corresponding TIMP-4 amino acid residues (E192V/D193Q). Together, this shows the importance of Glu(192) and Asp(193) in TIMP-2 binding to pro-MMP-2; the lack of these acidic residues in the TIMP-4 C-terminal tail, which reduces the stability of complex formation with the MMP-2 hemopexin C domain, probably precludes TIMP-4 from supporting the activation of pro-MMP-2.
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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 | 0.001 |
| 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".