Metal Substrate Selectivity and Mechanism of Transport in a Transmembrane Zn‐pump Revealed by <i>in‐vitro</i> Real‐time Transport
Bibliographic record
Abstract
P 1B ‐type ATPases are primary active transmembrane metal pumps conserved in all kingdoms of life that play a pivotal role in controlling cellular metal ion levels and homeostasis. In bacteria, they are responsible for selectively exporting essential and toxic metal ions across the cell membrane to maintain concentrations below toxic levels. They are critical virulence factors in pathogenic bacteria and allow selected microorganisms to survive in extreme metal‐rich environments. The P 1B‐2 ‐type Zn(II) ATPase pump subfamily possesses a wide promiscuity towards metal substrate translocation by plastic selection of Zn(II)/Cd(II)/Hg(II)/Pb(II) at their high‐affinity transmembrane metal binding site. Despite these substrates stimulate ATP‐hydrolysis, direct translocation of all these metals and their mechanism of transport remain to be demonstrated. In this work, we established a platform for purification and in‐vitro functional reconstitution in artificial phospholipid bilayer vesicles (proteoliposomes) of ZntA from Cupriavidus metallidurans to dissect the molecular mechanism of divalent metal ion translocation and substrate promiscuity towards first‐, second‐ and the third‐row transition metals. Metal‐dependent ATPase activity assays of Cm ΔZntA 166–794 in detergent micelles and proteoliposomes revealed stimulation of the ATPase activity by Pb(II), Zn(II), Cd(II) and Hg(II) substrates with the maximal velocities (V max ) following the order Pb(II) > Zn(II) > Cd(II) > Hg(II), confirming the successful functional reconstitution in lipid vesicles. By selecting and encapsulating divalent metal sensitive fluorescent probes in the proteoliposome lumen, we performed real‐time metal transport assays to reveal the ATP‐dependent translocation of Pb(II), Zn(II), Cd(II) and Hg(II) across the lipid bilayer, establishing that the translocation rates correlate to the order observed following ATP hydrolysis. Furthermore, to dissect the substrate transport mechanism, we encapsulated the pH sensitive fluorescent probe pyranine to demonstrated that, differently than other P‐type ATPases, translocation is not coupled to H + counter‐flux for all the M(II) substrates. By encapsulating the membrane potential sensitive fluorescent probe Oxonol‐VI we also revealed that, with all substrates, Zn‐pumps act as primary active electrogenic uniporters capable of generating a transmembrane potential. This work establishes a platform to dissect the mechanisms of translocation of various metal substrates in P‐type ATPases which is applicable to study other uncharacterized transporter families and to reveal the bioinorganic chemistry of metal ion transport at molecular level.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
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 | 0.001 | 0.000 |
| Open science | 0.001 | 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".