title: Molecular dissection of dual pseudosymmetric solute translocation pathways in human P-glycoprotein.
abstract: The human multispecific drug efflux transporter P-glycoprotein (P-gp) causes drug resistance and modulates the pharmacological profile of systemically administered medicines. It has arisen from a homodimeric ancestor by gene duplication. Crystal structures of mouse MDR1A indicate that P-gp shares the overall architecture with two homodimeric bacterial exporters, Sav1866 and MsbA, which have complete rotational symmetry. For ATP-binding cassette transporters, nucleotide binding occurs in two symmetric positions in the motor domains. Based on the homology with entirely symmetric half-transporters, the present study addressed the key question: can biochemical evidence for the existence of dual drug translocation pathways in the transmembrane domains of P-gp be found? P-gp was photolabeled with propafenone analogs, purified, and digested proteolytically, and peptide fragments were identified by high-resolution mass spectrometry. Labeling was assigned to two regions in the protein by projecting data into homology models. Subsequently, symmetric residue pairs in the putative translocation pathways were identified and replaced by site-directed mutagenesis. Transport assays corroborated the existence of two pseudosymmetric translocation pathways. Although rhodamine123 has a preference to take one path, verapamil, propafenones, and vinblastine preferentially use the other. Two major findings ensued from this study: the existence of two solute translocation pathways in P-gp as a reflection of evolutionary origin from a homodimeric ancestor and selective but not exclusive use of one of these pathways by different P-gp solutes. The pseudosymmetric behavior reconciles earlier kinetic and thermodynamic data, suggesting an alternative concept of drug transport by P-gp that will aid in understanding the off-target quantitative structure activity relationships of P-gp interacting drugs.
authors.affiliation: Institute of Medical Chemistry, Medical University of Vienna, Vienna, Austria.
authors.forename: Zahida
authors.initials: Z
authors.lastname: Parveen
authors.forename: Thomas
authors.initials: T
authors.lastname: Stockner
authors.forename: Caterina
authors.initials: C
authors.lastname: Bentele
authors.forename: Sandra
authors.initials: S
authors.lastname: Pferschy
authors.forename: Martin
authors.initials: M
authors.lastname: Kraupp
authors.forename: Michael
authors.initials: M
authors.lastname: Freissmuth
authors.forename: Gerhard F
authors.initials: GF
authors.lastname: Ecker
authors.forename: Peter
authors.initials: P
authors.lastname: Chiba
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D020112:Rhodamine 123 / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D020112:Rhodamine 123; C095122:multidrug resistance protein 3