Exploring P-Glycoprotein Substrate Access

Abstract

P-glycoprotein (P-gp), an ATP Binding Cassette transporter family membrane protein, extrudes mainly hydrophobic substrates in an ATP hydrolysis-dependant manner. Structurally, P-gp consists of two transmembrane domains (TMD) each comprised of 6 α-helices that together form the drug binding site, and two nucleotide binding domains (NBD) that via ATP binding and hydrolysis provide the energy for the conformational changes necessary to drive drug translocation.Due to the wide variety of substrates it extrudes, P-gp is one of the main causes of multidrug resistance in cancer and other diseases. As multidrug resistance becomes an ever increasingly important issue, drug development is dependant on knowing what constitutes the difference between a substrate and a non-substrate of P-gp. Although the chemical structure is likely to play a role in recognition within the binding site, it is equally important to understand how access to the binding site is controlled. Therefore more information about way in which substrates access the binding site is of key interest.In the absence of a human P-gp crystal structure, several human homology models were made from both eukaryotic and bacterial homologue crystal structures. The models were assessed for structural stability and conformational dynamics using molecular dynamics (MD) simulations. Following analysis, the homology model from the C. elegans template was chosen as the model for steered MD calculations to investigate possible substrate access pathways.The results, when combined with bilayer localisation NMR experiments and MD simulations, give novel insights into the factors that govern P-gp substrate specificity.