Structural Studies of Multi-Drug Resistance P-glycoprotein by Electron Microscopy
Multi-drug resistance P-glycoprotein (Pgp), a member of the ATP-binding cassette transporter superfamily, is associated with multi-drug resistance in cancer chemotherapy. In addition to cancer cells, Pgp can be found in organs or normal tissues, such as blood-tissue barriers, gastrointestinal tract or kidney, where it provides protection against toxic or xenobiotic materials. This indicates that Pgp plays a dual role: one in drug resistance in cancer and one in detoxification in normal cells, and it is important to investigate the mechanism of Pgp for understanding multi-drug resistance in cancer and for elucidating its physiological functions. P-glycoprotein is a single polypeptide consisting of -1280 amino acids. The protein contains two cytoplamic nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). It is believed that Pgp undergoes significant conformational changes during drug transport driven by ATP hydrolysis. In order to gain insight into the conformational changes, the structure of Pgp was studied by electron microscopy and image analysis. First, two-dimensional (2-D) crystals of Pgp were developed by the lipid-monolayer technique. A projection structure of Pgp in the absence of nucleotide or drug substrates was solved to 22-A resolution. The projection shows two closely interacting domains, thus providing evidence of a closed conformation of the NBDs. Second, a subtle rearrangement of the two domains has been observed in projection structures under various nucleotide conditions, showing the two domains shifted away to each other. This indicates that Pgp undergoes conformational changes during the ATP hydrolytic cycle. Yet, the conformational changes are subtle and may not induce a dramatic domain movement as proposed from crystal structures of bacterial lipid-A flippase (MsbA; Chang & Roth, 2001). Third, three-dimensional (3-D) structures of membrane-bound Pgp in the nucleotide-free and ATPyS-bound states were obtained by 3-D reconstruction from tilted images of negatively stained specimen. Based on the projections and 3-D models, although at a modest 22-A resolution, a refined mechanism is presented that describes the domain-domain interaction during the catalytic cycle. This study illustrate the first detailed structural information of lipid-bilayer-bound Pgp showing a conformational change in the functional domains due to ATP binding to the NBDs.