Received for publication September 7, 2004.
Revised October 26, 2004.
Accepted for publication October 27, 2004.

P-glycoprotein substrate binding domains are located at the transmembrane domain : transmembrane domain interfaces - A combined photoaffinity labeling - protein homology modeling approach

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 ADMET (absorption, distribution, metabolism, excretion and toxicity) profiling in drug development. A set of propafenone-type substrate photoaffinity ligands has been used in this study in conjunction with MALDI-TOF mass-spectrometry to define the substrate-binding-domain(s) of P-gp in more detail. Highest labeling was observed in transmembrane segments 3, 5, 8 and 11. A homology model for P-gp was generated based on the dimeric crystal structure of Vibrio cholerae MsbA (Vc-MsbA), an essential lipid transporter. Subsequently, 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 TMD:TMD interfaces formed between the amino- and carboxy terminal half of P-gp. These interfaces are formed by 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.

Key words: MDR/p-Glycoprotein, Thermodynamic and kinetic processes and modeling, Mass Spectroscopy, Resistance