Combining in Vitro with in Silico Studies to Obtain Insights into Substrate Releasing State of the Multidrug Resistance Protein P-Glycoprotein

Abstract

The human genome contains 48 ATP-Binding Cassette (ABC) proteins. We focus on the multidrug resistance transporter P-glycoprotein that transports an extraordinarily diverse range of structurally unrelated drugs, xenobiotics and endogenous substrates. P-glycoprotein is expressed at barrier tissues including the blood-brain-barrier, the intestine, kidney, liver and macrophages. Cancer cells acquire resistance to chemotherapy when expressing P-glycoprotein. It is well established that ATP binding and its subsequent hydrolysis drives the transport process in all ABC transporters. In contrast, most details regarding the mechanism of substrate recognition, uptake and binding to P-glycoprotein, and the mechanism by which substrate binding in the transmembrane domains is coupled to ATP hydrolysis in the nucleotide binding domains remain unknown.The bacterial homologue of P-gp, Sav1866 (Staphylococcus aureus), was the first ABC exporter crystallized. The same fold was later observed in other transporters of the ABCB family, suggesting a conserved architecture across the ABCB exporter family. Although ABC exporters have now been crystallized in several conformations, uncertainty remains regarding the physiological conformation of these structures. None of the crystal structures is fully compatible with all biochemical evidence.We combined modeling with experiments to address these issues. Homology modeling and MD simulations were used to determine the equilibrium conformation of ATP-bound P-glycoprotein in a membrane environment. In contrast to the conformations observed in crystal structures, the wing shape structure is unstable in the membrane environment. The conformation observed by MD simulations is devoid of the wing-shape, but in agreement with the bulk of the biochemical data.Acknowledgements: The research was funded by the Austrian Science Fund (FWF): P23319-B11.