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Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET
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Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition
Pratiti Ghosh1, Karobi Moitra, Nazli Maki
1Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, F. Edward Hébert School of Medicine, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA.
Archives of Biochemistry and Biophysics
|April 21, 2006
Summary
Certain thioxanthene modulators, like flupentixol, alter P-glycoprotein (Pgp) conformation through an allosteric site. This change protects Pgp from digestion, mimicking nucleotide binding effects and impacting drug transport.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Human P-glycoprotein (Pgp, ABCB1) is a crucial drug transporter whose function can be modulated by various agents.
- Understanding Pgp modulation mechanisms is vital for optimizing drug therapies and overcoming multidrug resistance.
Purpose of the Study:
- To investigate the conformational changes induced by thioxanthene-based Pgp modulators with an allosteric mode of action.
- To elucidate the role of the allosteric site in Pgp modulation and its effect on proteolytic digestion susceptibility.
Main Methods:
- Utilizing trypsin digestion assays to assess conformational changes in Pgp fragments.
- Employing specific antibodies to identify Pgp fragments and analyzing the impact of flupentixol isomers and mutants (Pgp F983A).
- Comparing the effects of ATP, ATPgammaS, ADP-vanadate, vinblastine, and cyclosporin A on Pgp conformation.
Main Results:
- Flupentixol isomers (cis and trans) protected an 80 kDa Pgp fragment from trypsin digestion, indicating a conformational shift.
- This protective effect was abolished in the Pgp F983A mutant, confirming the involvement of the allosteric site.
- ATP, ATPgammaS, and ADP-vanadate also conferred protection, suggesting mimicry of catalytic transition states, unlike substrate vinblastine or competitive modulator cyclosporin A.
Conclusions:
- Allosteric interaction of flupentixols with Pgp induces conformational changes that resemble catalytic intermediates.
- These conformational changes, distinct from substrate or competitive modulator interactions, are key to allosteric inhibition of Pgp-mediated drug transport.
- The findings provide insights into the allosteric regulation of Pgp, potentially guiding the development of novel Pgp modulators.
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