title: Functional role of the linker region in purified human P-glycoprotein.
abstract: Human P-glycoprotein (P-gp), which conveys multidrug resistance, is an ATP-dependent drug efflux pump that transports a wide variety of structurally unrelated compounds out of cells. P-gp possesses a 'linker region' of approximately 75 amino acids that connects two homologous halves, each of which contain a transmembrane domain followed by a nucleotide-binding domain. To investigate the role of the linker region, purified human P-gp was cleaved by proteases at the linker region and then compared with native P-gp. Based on a verapamil-stimulated ATP hydrolase assay, size-exclusion chromatography analysis and a thermo-stability assay, cleavage of the P-gp linker did not directly affect the preservation of the overall structure or the catalytic process in ATP hydrolysis. However, linker cleavage increased the k(cat) values both with substrate (k(sub)) and without substrate (k(basal)), but decreased the k(sub)/k(basal) values of all 10 tested substrates. The former result indicates that cleaving the linker activates P-gp, while the latter result suggests that the linker region maintains the tightness of coupling between the ATP hydrolase reaction and substrate recognition. Inspection of structures of the P-gp homolog, MsbA, suggests that linker-cleaved P-gp has increased ATP hydrolase activity because the linker interferes with a conformational change that accompanies the ATP hydrolase reaction. Moreover, linker cleavage affected the specificity constants [k(sub)/K(m(D))] for some substrates (i.e. linker cleavage probably shifts the substrate specificity profile of P-gp). Thus, this result also suggests that the linker region regulates the inherent substrate specificity of P-gp.
authors.affiliation: Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Sato
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kodan
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Kimura
authors.forename: Kazumitsu
authors.initials: K
authors.lastname: Ueda
authors.forename: Toru
authors.initials: T
authors.lastname: Nakatsu
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kato
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000378:metabolism; D018432:Drug Resistance, Multiple / Q000502:physiology; D006801:Humans; D006868:Hydrolysis; D008081:Liposomes / Q000378:metabolism; D008823:Micelles; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D008081:Liposomes; D008823:Micelles; C079726:MsbA protein, Bacteria; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin; D000251:Adenosine Triphosphatases