Refined structures of mouse P-glycoprotein.

Area of Science:

• Structural Biology
• Biochemistry
• Molecular Biology

Background:

• The initial mouse P-glycoprotein (Pgp) structure model showed discrepancies with the Caenorhabditis elegans Pgp structure.
• These discrepancies suggested potential inaccuracies in the original mouse Pgp model.

Purpose of the Study:

• To generate a more accurate experimental electron density map of the original mouse Pgp.
• To refine the mouse Pgp structure and identify previously unrecognized drug-binding residues.

Main Methods:

• Single-wavelength anomalous dispersion (SAD) phasing was used to generate an experimental electron density map at 3.8 Å resolution.
• De novo model building and refinement were performed, with validation using mercury labeling and anomalous Fourier density.
• Protein geometry was assessed using Ramachandran analysis.

Main Results:

• The refined mouse Pgp model achieved 3.8 Å resolution with improved R-factors (Rwork=21.2%, Rfree=26.6%).
• Protein geometry significantly improved, with ~95% of residues in favorable Ramachandran regions, compared to 57% in the original model.
• Corrections to transmembrane helix registry, particularly TM4 and TM5, revealed new drug-binding residues and a drug entry portal.

Conclusions:

• The corrected mouse Pgp structure provides a more accurate representation of the drug-binding and translocation mechanisms.
• The drug translocation pathway is highly conserved between mouse and human Pgp, suggesting a shared mechanism for polyspecific substrate recognition.