title: Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle.
abstract: P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.
authors.affiliation: Department of Biomolecular Sciences, UMIST, Manchester M60 1QD, UK.
authors.forename: M F
authors.initials: MF
authors.lastname: Rosenberg
authors.forename: G
authors.initials: G
authors.lastname: Velarde
authors.forename: R C
authors.initials: RC
authors.lastname: Ford
authors.forename: C
authors.initials: C
authors.lastname: Martin
authors.forename: G
authors.initials: G
authors.lastname: Berridge
authors.forename: I D
authors.initials: ID
authors.lastname: Kerr
authors.forename: R
authors.initials: R
authors.lastname: Callaghan
authors.forename: A
authors.initials: A
authors.lastname: Schmidlin
authors.forename: C
authors.initials: C
authors.lastname: Wooding
authors.forename: K J
authors.initials: KJ
authors.lastname: Linton
authors.forename: C F
authors.initials: CF
authors.lastname: Higgins
mesh_terms: D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002384:Catalysis; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D020285:Cryoelectron Microscopy; D003460:Crystallization; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D018432:Drug Resistance, Multiple; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006868:Hydrolysis; D007313:Insecta; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D020245:p-Chloromercuribenzoic Acid; D002731:4-Chloromercuribenzenesulfonate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D003545:Cysteine