Stable Structural Analog of Ca2+-ATPase ADP-insensitive Phosphoenzyme with Occluded Ca2+ Formed by Elongation of A-domain/M1′-linker and Beryllium Fluoride Binding
Bibliographic record
Abstract
We have developed a stable analog for the ADP-insensitive phosphoenzyme intermediate with two occluded Ca(2+) at the transport sites (E2PCa(2)) of sarcoplasmic reticulum Ca(2+)-ATPase. This is normally a transient intermediate state during phosphoenzyme isomerization from the ADP-sensitive to ADP-insensitive form and Ca(2+) deocclusion/release to the lumen; E1PCa(2) --> E2PCa(2) --> E2P + 2Ca(2+). Stabilization was achieved by elongation of the Glu(40)-Ser(48) loop linking the Actuator domain and M1 (1st transmembrane helix) with four glycine insertions at Gly(46)/Lys(47) and by binding of beryllium fluoride (BeF(x)) to the phosphorylation site of the Ca(2+)-bound ATPase (E1Ca(2)). The complex E2Ca(2)xBeF(3)(-) was also produced by lumenal Ca(2+) binding to E2xBeF(3)(-) (E2P ground state analog) of the elongated linker mutant. The complex was stable for at least 1 week at 25 degrees C. Only BeF(x), but not AlF(x) or MgF(x), produced the E2PCa(2) structural analog. Complex formation required binding of Mg(2+), Mn(2+), or Ca(2+) at the catalytic Mg(2+) site. Results reveal that the phosphorylation product E1PCa(2) and the E2P ground state (but not the transition states) become competent to produce the E2PCa(2) transient state during forward and reverse phosphoenzyme isomerization. Thus, isomerization and lumenal Ca(2+) release processes are strictly coupled with the formation of the acylphosphate covalent bond at the catalytic site. Results also demonstrate the critical structural roles of the Glu(40)-Ser(48) linker and of Mg(2+) at the catalytic site in these processes.
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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 | 0.000 | 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) | 0.000 | 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".