How Does the Multidrug Resistance P-glycoprotein Recognise Multiple Drugs

Abstract

P-glycoprotein, a multidrug resistance transporter, is able to bind and transport a wide range of structurally and functionally unrelated compounds. The biochemical and pharmacological data attributes this poly-specificity to the presence of at least four pharmacologically distinct binding sites which are spatially distributed. However, the pharmacophore as well as the mechanism of substrate transport remains elusive. Current investigation expands the understanding of two of these binding sites, binding site for the anticancer drug vinblastine at the lipid protein interface and the binding site for fluorescent probe rhodamine123, a known substrate of P-glycoprotein, near central cavity. The aim was to explore how these two substrates bind and are translocated. A series of mutations was generated in positions proximal to previously defined drug-interacting residues on P-glycoprotein. The protein was purified and reconstituted into styrene-maleic acid lipid particles (SMALPs) to measure the apparent drug binding constant or into liposomes for assessment of drug-stimulated ATP hydrolysis. The combination of biochemical and structural data generated suggests that initial binding may occur in the central cavity or at the lipid-protein interface. The biochemical data were reconciled with structural models of P-glycoprotein using molecular docking. The data indicated that the binding of rhodamine 123 occurred predominantly within the central cavity of P-glycoprotein. In contrast, the significantly more hydrophobic vinblastine bound to both the lipid-protein interface and within the central cavity. The data suggest that the initial interaction of vinca alkaloids with P-glycoprotein occurs at the lipid interface followed by internalisation into the central cavity, which also provides the transport conduit. This model is supported by recent structural observations and early biophysical and cross-linking approaches. Moreover, the proposed model illustrates that the broad substrate profile for P-glycoprotein is underpinned by a combination of multiple initial interaction sites and an accommodating transport conduit. Show more