The Human Breast Cancer Resistance Protein (BCRP/ABCG2) Shows Conformational Changes with Mitoxantrone

BCRP/ABCG2 mediates efflux of drugs and xenobiotics. BCRP was expressed in Pichia pastoris, purified to > 90% homogeneity, and subjected to two-dimensional (2D) crystallization. The 2D crystals showed a p12(1) symmetry and projection maps were determined at 5 angstrom resolution by cryo-electron microscopy. Two crystal forms with and without mitoxantrone were observed with unit cell dimensions of a = 55.4 angstrom, b = 81.4 angstrom, gamma = 89.8 degrees, and a = 57.3 angstrom, b = 88.0 angstrom, gamma = 89.7 degrees, respectively. The projection map without mitoxantrone revealed an asymmetric structure with ring-shaped density features probably corresponding to a bundle of transmembrane a helices, and appeared more open and less symmetric than the map with mitroxantrone. The open and closed inward-facing forms of BCRP were generated by homology modeling, representing the substrate-free and substrate-bound conformations in the absence of nucleotide, respectively. These models are consistent with the experimentally observed conformational change upon substrate binding.