Drug-Binding to Distinct Sites of the Multidrug Exporter P-Glycoprotein

P-glycoprotein (Pgp) exports hundreds of chemically unrelated, hydrophobic compounds out cells. Since Pgp can greatly affect bioavailability, pharmacokinetics and efficacy therapeutic drugs, there is great interest in understanding the mechanism by which drugs are extruded. an ABC transporter with two nucleotide binding domains (NBDs) that bind hydrolyze ATP, leading to extensive conformational changes across transmembrane (TMDs) result substrate translocation cell membrane. Despite decades biochemical/biophysical studies, NBDs control these changes, how this linked drug TMDs, ultimately export still controversial. Our recent X-ray structures identified aromatic amino acids contribute different inhibitors drug-binding site. study, we test hypothesis subsets residues within defined subpockets TMDs protein. general approach introduce tryptophans (Trps) at strategic positions order monitor binding. These Trps were introduced on background a new fully functional low-Trp retains three native conformationally sensitive cytoplasmic domains, or onto Trp-less Pgp. Using purified protein reconstituted lipid bilayer nanodiscs, fluorescence such as quenching, resonance energy transfer (FRET), present first results interactions prototypical substrates, cyclopeptides QZ-Val QZ-Phe occupy structurally distinct sites, well nicardipine zosuquidar. The impact mechanisms will be discussed.