UC San Diego

Multidrug transporters are membrane-imbedded proteins which mediate the traversal of molecules across the cell membrane. Of particular interest herein, the multidrug and toxic compound extrusion (MATE) family of secondary transporters play critical roles in plant metabolite transport, bacterial multidrug resistance (MDR), and mammalian renal function. The outward (extracellular) facing 3.65Å structure reported was the first for this family of transporters, and additionally revealed a site which may serve to bind cations and provide the "power stroke" during the transport cycle. This work also implemented a novel application of a technique to validate the topology and registration of a moderate resolution structure. The adenosine triphosphate (ATP)-binding cassette (ABC) family of primary transporters are perhaps best exemplified by p-glycoprotein (P-gp), possibly the most studied of all membrane transporters. In humans, P-gp is expressed throughout the body, including the gut lumen, kidneys, blood-brain barrier, and functions as a ubiquitous molecular "gate-keeper" in the recognition and expulsion of substances from the cytoplasm and intermembrane leaflet. As such, P-gp plays well- categorized roles in mediating the bioavailability of pharmaceuticals, and it's overexpression in some forms of cancer has been linked to MDR. Previous to this work, a 3.9Å structure of mouse P-gp had been elucidated. However, a more detailed structural understanding of the means by which P-gp recognizes and expels molecules has remained nebulous. The work described here expands on previous knowledge by generating a topologically validated novel structure of mouse P-gp extending to 3.4Å, the discovery of a novel epitope binding site on the cytoplasmic nucleotide-binding domain (NBD) face of this molecule, and the first structure-activity relationship study (SAR) for this protein utilizing a rationally engineered series of ligands in order to probe the transporter:substrate relationship