title: 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 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.
authors.affiliation: Institute of Medical Chemistry, Medical University of Vienna, Waehringerstrasse 10, A-1090 Vienna, Austria.
authors.forename: Karin
authors.initials: K
authors.lastname: Pleban
authors.forename: Stephan
authors.initials: S
authors.lastname: Kopp
authors.forename: Edina
authors.initials: E
authors.lastname: Csaszar
authors.forename: Michael
authors.initials: M
authors.lastname: Peer
authors.forename: Thomas
authors.initials: T
authors.lastname: Hrebicek
authors.forename: Andreas
authors.initials: A
authors.lastname: Rizzi
authors.forename: Gerhard F
authors.initials: GF
authors.lastname: Ecker
authors.forename: Peter
authors.initials: P
authors.lastname: Chiba
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D007313:Insecta; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000737:chemistry / Q000378:metabolism*; D011405:Propafenone / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013499:Surface Properties
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011405:Propafenone