Purification and 3D structural analysis of oligomeric human multidrug transporter ABCG2

被引:101
作者
McDevitt, Christopher A.
Collins, Richard F.
Conway, Michael
Modok, Szabolcs
Storm, Janet
Kerr, Ian D.
Ford, Robert C.
Callaghan, Richard [1 ]
机构
[1] Univ Oxford, John Radcliffe Hosp, Nuffield Dept Clin Lab Sci, Oxford OX3 9DU, England
[2] Univ Manchester, Fac Life Sci, Manchester M60 1QD, Lancs, England
[3] Univ Nottingham, Ctr Biochem & Cell Biol, Sch Biomed Sci, Queens Med Ctr, Nottingham NG7 2RD, England
基金
英国医学研究理事会;
关键词
D O I
10.1016/j.str.2006.08.014
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to 18 angstrom. Single-particle analysis revealed that the complex forms a tetrameric complex (similar to 180 angstrom in diameter x similar to 140 angstrom high) with an aqueous central region. We interpret the tetrameric structure as comprising four homodimeric ABCG2(R482G) complexes.
引用
收藏
页码:1623 / 1632
页数:10
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