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Conformational changes of P-glycoprotein by nucleotide binding
1Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641, USA.
The Biochemical Journal
|February 7, 1998
Summary
P-glycoprotein (Pgp) undergoes conformational changes during its ATP hydrolysis cycle, influenced by nucleotide binding. These changes are crucial for coupling ATP use to drug transport, potentially overcoming multidrug resistance.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Biology
Background:
- P-glycoprotein (Pgp) is a key efflux pump in multidrug resistance (MDR).
- Pgp belongs to the ATP-binding cassette (ABC) superfamily and functions as a transport ATPase.
- Conformational changes in Pgp are hypothesized to be central to its catalytic cycle.
Purpose of the Study:
- To investigate the conformational changes of Pgp during its catalytic cycle.
- To identify distinct Pgp conformations induced by nucleotide ligands and inhibitors.
- To elucidate the relationship between Pgp's ATPase activity, ligand binding, and drug transport.
Main Methods:
- Limited proteolysis was employed to probe Pgp conformation.
- Inside-out membrane vesicles from SKOV/VLB cells expressing human Pgp were used.
- Proteolysis patterns were analyzed in the presence of MgATP, Mg-p[NH]ppA, and MgADP.
Main Results:
- Nucleotide binding (MgATP, Mg-p[NH]ppA, MgADP) altered Pgp's proteolysis profile, indicating conformational shifts.
- The loop connecting transmembrane regions TM8 and TM9 was sensitive to nucleotide binding.
- Four distinct conformational states of Pgp were stabilized by various ligands and inhibitors.
Conclusions:
- Nucleotide binding and/or hydrolysis induce significant conformational changes in Pgp.
- These conformational changes are directly linked to Pgp's ATPase activity.
- A model is proposed where cycling through four states couples Pgp's MgATP hydrolysis to drug transport, offering insights into overcoming MDR.