Structural Studies of Multidrug Resistance Protein 1 Using “Almost” Cysless Template
Bibliographic record
Abstract
Multidrug resistance protein, MRP1 (ABCC1) is a broad-spectrum ATP-binding cassette transporter that plays a major role in defense against dietary and environmental toxicants, in addition to contributing toward multidrug resistance of certain types of malignancy. Elucidating the molecular structure of hMRP1 is key to determining its mechanism of substrate recognition and transport. Here, we report the first successful attempt using cysteine-scanning mutagenesis coupled with cross-linking studies to probe the structure of hMRP1 in its native environment of the cell membrane or in membrane vesicles. We have established that an active 3Cys ΔMRP1 (MRP204–1531) mutant, described in previous studies from our laboratory, is a suitable template with which to generate single- and double-cysteine mutants for performing cysteine mutagenesis studies. We have now used 3Cys ΔMRP1 to probe the arrangement of several TM segments, as well as the location of individual amino acids in these regions. Cysteine residues were introduced into TMs 8, 14, 15, and 16 of 3Cys ΔMRP1. The mutants were then subjected to chemical cross-linking analyses, and cross-linking was detected between the following cysteine pairs: Cys388 (TM7) and I1193C (TM16); Cys388 (TM7) and E1144C (TM15); R433C (TM8) and E1144C (TM15); and R433C (TM8) and T1082C (TM14). The aqueous accessibility of these residues and the possible implications of the differences between the open and closed states of the protein are also discussed. Moreover, using competition experiments involving a well characterized substrate and a cross-linking reagent for probing the Cys388/ I1193C mutant, we have defined these amino acid positions as a component of the potential site for estrone sulfate binding.
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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".