Purification and 3D Structural Analysis of Oligomeric Human Multidrug Transporter ABCG2

Structure. 2006 Nov;14(11):1623-32. doi: 10.1016/j.str.2006.08.014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily G, Member 2
  • ATP-Binding Cassette Transporters / chemistry*
  • ATP-Binding Cassette Transporters / isolation & purification*
  • Animals
  • Cell Membrane / metabolism
  • Chromatography / methods
  • Cryoelectron Microscopy
  • Detergents / chemistry
  • Detergents / pharmacology
  • Dimerization
  • Humans
  • Image Processing, Computer-Assisted
  • Imaging, Three-Dimensional
  • Insecta
  • Neoplasm Proteins / chemistry*
  • Neoplasm Proteins / isolation & purification*
  • Protein Conformation
  • Protein Isoforms
  • Protein Structure, Tertiary

Substances

  • ABCG2 protein, human
  • ATP Binding Cassette Transporter, Subfamily G, Member 2
  • ATP-Binding Cassette Transporters
  • Detergents
  • Neoplasm Proteins
  • Protein Isoforms