title: Purification and 3D structural analysis of oligomeric human multidrug transporter ABCG2.
abstract: ABCG2 is a multidrug efflux pump associated with resistance of cancer cells to a plethora of unrelated drugs. ABCG2 is a "half-transporter," and previous studies have indicated that it forms homodimers and higher oligomeric species. In this manuscript, electron microscopic structural analysis directly addressed this issue. An N-terminal hexahistidine-tagged ABCG2(R482G) isoform was expressed to high levels in insect cells. An extensive detergent screen was employed to effect extraction of ABCG2(R482G) from membranes and identified only the fos-choline detergents as efficient. Soluble protein was purified to >95% homogeneity by a three-step procedure while retaining the ability to bind substrates. Cryonegative stain electron microscopy of purified ABCG2(R482G) provided 3D structural data at a resolution of approximately 18 A. Single-particle analysis revealed that the complex forms a tetrameric complex ( approximately 180 A in diameter x approximately 140 A high) with an aqueous central region. We interpret the tetrameric structure as comprising four homodimeric ABCG2(R482G) complexes.
authors.affiliation: Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DU, United Kingdom.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: McDevitt
authors.forename: Richard F
authors.initials: RF
authors.lastname: Collins
authors.forename: Michael
authors.initials: M
authors.lastname: Conway
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Modok
authors.forename: Janet
authors.initials: J
authors.lastname: Storm
authors.forename: Ian D
authors.initials: ID
authors.lastname: Kerr
authors.forename: Robert C
authors.initials: RC
authors.lastname: Ford
authors.forename: Richard
authors.initials: R
authors.lastname: Callaghan
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000302:isolation & purification*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D020285:Cryoelectron Microscopy; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology; D019281:Dimerization; D006801:Humans; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D007313:Insecta; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D003902:Detergents; D009363:Neoplasm Proteins; D020033:Protein Isoforms