P-glycoprotein transport cycle: 'cross-talk' between multiple binding sites and the catalytic domains

Martin, Catherine Anne (2001). P-glycoprotein transport cycle: 'cross-talk' between multiple binding sites and the catalytic domains. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000fd02

Abstract

Elucidation of the mechanism by which P-gp can interact with multiple drugs, and harness the energy from hydrolysis of ATP to mediate transbilayer movement of drug, has been the goal of this thesis. Using radioligand binding studies, four distinct binding sites for drug have been identified on P-gp, explaining the molecular basis of its broad ‘substrate’ specificity. Two of the sites have been classified as transport sites and have been assigned the sites of interaction for vinblastine and paclitaxel respectively. A third site identified was capable of binding both drugs that are transported (Hoechst 33342) by P-gp, and those that are modulatory only (XR9576 and XR9051). The fourth binding site has been classified as a modulatory site, and has been assigned to the modulators nicardipine and GF120918. The existence of a negative heterotropic allosteric network between the sites, suggests that P-gp does not simultaneously bind multiple drugs. In order for transport of drug to occur, the drug binding site must undergo ‘re-orientation’, switching from a high to a low affinity conformation. This transition must be associated with ATP hydrolysis, given the ATP dependency of P-gp transport function. Therefore, binding of vinblastine was measured at discrete steps of the catalytic cycle of P-gp, to ascertain when changes are wrought on the drug binding site. Binding of ATP, prior to hydrolysis, triggered a shift in the high affinity site to a single class of low affinity site. The low affinity conformation for the vinblastine binding site persisted immediately post-hydrolysis of nucleotide. Release of Pi from the P-gp.Mg.ADP.Pi complex, fully restored the drug binding site to high affinity once more. This has provided the first evidence to show that the binding of ATP is sufficient to instigate a transport event. Hydrolysis of ATP is most likely necessary to restore the drug binding site to high affinity in order to complete a transport cycle.