Tracing the substrate translocation mechanism in P-glycoprotein.

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter crucial for drug pharmacokinetics and cancer multidrug resistance.
• Previous studies elucidated Pgp's substrate binding but not the translocation mechanism across membranes.

Purpose of the Study:

• To elucidate the mechanism of substrate translocation through P-glycoprotein.
• To identify key structural elements and conformational changes involved in Pgp-mediated transport.

Main Methods:

• Cryo-electron microscopy (cryoEM) was used to determine structures of Pgp with a covalently attached cyclic substrate in different states.
• Molecular dynamics simulations were employed to trace substrate passage and analyze conformational changes.
• Site-directed mutagenesis of key residues was performed to assess functional impact.

Main Results:

• Multiple cryoEM structures captured Pgp in inward- and outward-facing states during substrate translocation.
• Conformational changes in transmembrane helix 1 (TM1), including a glycine 72 break, were identified as critical regulators of transport.
• A glycine 72 mutation significantly impaired Pgp's in vivo drug transport function.
• Substrate exit was observed without a fully open outward-facing conformation, suggesting a non-canonical efflux model.

Conclusions:

• The study reveals a novel mechanism for P-glycoprotein substrate transport, highlighting the regulatory role of TM1 conformational dynamics.
• The findings challenge the traditional model of ABC transporter efflux and provide critical insights into Pgp function.
• This work identifies potential targets for developing new strategies to overcome Pgp-mediated drug resistance.