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

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 ABCG2R482G isoform was expressed to high levels in insect cells. An extensive detergent screen was employed to effect extraction of ABCG2R482G 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 ABCG2R482G provided 3D structural data at a resolution of 18 Å. Single-particle analysis revealed that the complex forms a tetrameric complex (180 Å in diameter × 140 Å high) with an aqueous central region. We interpret the tetrameric structure as comprising four homodimeric ABCG2R482G complexes.Christopher A. McDevitt, Richard F. Collins, Michael Conway, Szabolcs Modok, Janet Storm, Ian D. Kerr, Robert C. Ford and Richard Callagha