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ORIGINAL ARTICLE

P-Glycoprotein Substrate Binding Domains Are Located at the Transmembrane Domain/Transmembrane Domain Interfaces: A Combined Photoaffinity Labeling-Protein Homology Modeling Approach

Institute of Medical Chemistry (K.P., S.K., M.P., P.C.), Medical University of Vienna, Department of Pharmaceutical Chemistry (K.P., G.F.E.), Institute of Analytical Chemistry (T.H., A.R.), and the Mass Spectrometry Facility, Max F. Perutz Laboratories, University Departments at the Vienna Biocenter (E.C.), University of Vienna, Vienna, Austria

Abstract

P-glycoprotein (P-gp) is an energy-dependent multidrug efflux pump conferring resistance to cancer chemotherapy. Characterization of the mechanism of drug transport at a molecular level represents an important prerequisite for the design of pump inhibitors, which resensitize cancer cells to standard chemotherapy. In addition, P-glycoprotein plays an important role for early absorption, distribution, metabolism, excretion, and toxicity profiling in drug development. A set of propafenonetype substrate photoaffinity ligands has been used in this study in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to define the substrate binding domain(s) of P-gp in more detail. The highest labeling was observed in transmembrane segments 3, 5, 8, and 11. A homology model for P-gp was generated on the basis of the dimeric crystal structure of Vibrio cholerae MsbA, an essential lipid transporter. Thereafter, the labeling pattern was projected onto the 3D atomic-detail model of P-gp to allow a visualization of the binding domain(s). Labeling is predicted by the model to occur at the two transmembrane domain/transmembrane domain interfaces formed between the amino- and carboxyl-terminal half of P-gp. These interfaces are formed by transmembrane (TM) segments 3 and 11 on one hand and TM segments 5 and 8 on the other hand. Available data on LmrA and AcrB, two bacterial multidrug efflux pumps, suggest that binding at domain interfaces may be a general feature of polyspecific drug efflux pumps.

Address correspondence to: Dr. Peter Chiba, Institute of Medical Chemistry, Medical University of Vienna, Waehringerstrasse 10, A-1090 Vienna, Austria. E-mail: peter.chiba{at}meduniwien.ac.at

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