P-glycoprotein (Pgp; also called multidrug resistance protein) is found in the plasma membrane of higher eukaryotes where it is responsible for ATP-hydrolysis-driven export of hydrophobic molecules. In animals, Pgp plays an important role in excretion of and protection from environmental toxins. When expressed in the plasma membrane of human cancer cells, Pgp can lead to failure of chemotherapy by preventing the hydrophobic chemotherapeutic drugs from reaching their targets inside the cells. Pgp is a member of the superfamily of ATP binding cassette (ABC) transporter proteins. ABC transporters consist typically of four domains, two nucleotide binding domains (NBDs) located in the cytoplasm and two trans-membrane domains (TMDs) responsible for drug binding and transport. Despite its important role in human disease, relatively little is known about the structure of Pgp. We are using electron microscopy of two-dimensional crystals to study the structure of Pgp. The immediate goals of this proposal are 1) to visualize the structural changes Pgp is undergoing during the catalytic cycle, 2) to calculate a three dimensional model of Pgp trapped at the different steps during ATP hydrolysis and drug transport and 3) to optimize the conditions under which we currently generate two dimensional crystals of Pgp. The structural studies will be conducted with Pgp crystallized in its native environment, the lipid bilayer. A three dimensional model of Pgp will serve as a basis for resolving the structural changes which Pgp is expected to undergo during the drug transport cycle. Understanding these structural changes might ultimately aid in the design for specific inhibitors for the protein in order to be able to regulate the activity of Pgp for a more effective chemotherapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA100246-01A1
Application #
6823886
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Lees, Robert G
Project Start
2004-07-01
Project End
2009-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
1
Fiscal Year
2004
Total Cost
$179,553
Indirect Cost
Name
University of California Riverside
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
627797426
City
Riverside
State
CA
Country
United States
Zip Code
92521
Zarrabi, Nawid; Ernst, Stefan; Verhalen, Brandy et al. (2014) Analyzing conformational dynamics of single P-glycoprotein transporters by Förster resonance energy transfer using hidden Markov models. Methods 66:168-79
Wen, Po-Chao; Verhalen, Brandy; Wilkens, Stephan et al. (2013) On the origin of large flexibility of P-glycoprotein in the inward-facing state. J Biol Chem 288:19211-20
Verhalen, Brandy; Ernst, Stefan; Börsch, Michael et al. (2012) Dynamic ligand-induced conformational rearrangements in P-glycoprotein as probed by fluorescence resonance energy transfer spectroscopy. J Biol Chem 287:1112-27
Verhalen, Brandy; Wilkens, Stephan (2011) P-glycoprotein retains drug-stimulated ATPase activity upon covalent linkage of the two nucleotide binding domains at their C-terminal ends. J Biol Chem 286:10476-82
Pearce, Margaret M P; Wormer, Duncan B; Wilkens, Stephan et al. (2009) An endoplasmic reticulum (ER) membrane complex composed of SPFH1 and SPFH2 mediates the ER-associated degradation of inositol 1,4,5-trisphosphate receptors. J Biol Chem 284:10433-45
Kish-Trier, Erik; Wilkens, Stephan (2009) Domain architecture of the stator complex of the A1A0-ATP synthase from Thermoplasma acidophilum. J Biol Chem 284:12031-40
Zhang, Zhenyu; Zheng, Yesha; Mazon, Hortense et al. (2008) Structure of the yeast vacuolar ATPase. J Biol Chem 283:35983-95
Kitagawa, Norton; Mazon, Hortense; Heck, Albert J R et al. (2008) Stoichiometry of the peripheral stalk subunits E and G of yeast V1-ATPase determined by mass spectrometry. J Biol Chem 283:3329-37
Lee, Jyh-Yeuan; Urbatsch, Ina L; Senior, Alan E et al. (2008) Nucleotide-induced structural changes in P-glycoprotein observed by electron microscopy. J Biol Chem 283:5769-79
Kish-Trier, Erik; Briere, Lee-Ann K; Dunn, Stanley D et al. (2008) The stator complex of the A1A0-ATP synthase--structural characterization of the E and H subunits. J Mol Biol 375:673-85

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