Volume 11, Issue 4, Pages 1093-1100 (April 2003) Structural Basis for Bile Acid Binding and Activation of the Nuclear Receptor FXR  Li-Zhi Mi, Srikripa Devarakonda, Joel M. Harp, Qing Han, Roberto Pellicciari, Timothy M. Willson, Sepideh Khorasanizadeh, Fraydoon Rastinejad  Molecular Cell  Volume 11, Issue 4, Pages 1093-1100 (April 2003) DOI: 10.1016/S1097-2765(03)00112-6 Copyright © 2003 Cell Press Terms and Conditions

Figure 1 Overall Views of FXR and Its Ligand Interactions (A) Perpendicular views of the FXR LBD (purple) from complex b of the 6ECDCA complex. Helix 12 of the receptor is shown in yellow, the GRIP-1 peptides are shown in blue and red, and 6ECDCA is shown in green. (B) Stereo view of a simulated annealing omit electron density (Fo-Fc) map showing the bound 6ECDCA. The ligand was excluded in map calculation. (C) Schematic representation of the FXR/6ECDCA interactions. Dotted red lines indicate hydrogen bonds; dotted blue lines indicate van der Waals contacts. Molecular Cell 2003 11, 1093-1100DOI: (10.1016/S1097-2765(03)00112-6) Copyright © 2003 Cell Press Terms and Conditions

Figure 2 The Activation Mechanism of FXR (A) 6ECDCA and 3-deoxyCDCA interactions with the activation switch of FXR. Shown is the superposition of their two crystal structures in the vicinity of the ligand and residues His444 and Trp466. (B) The binding of GRIP-1 NID-3 coactivator peptide to FXR as a function of increasing 3-deoxyCDCA (black triangles) and CDCA (red circles). Molecular Cell 2003 11, 1093-1100DOI: (10.1016/S1097-2765(03)00112-6) Copyright © 2003 Cell Press Terms and Conditions

Figure 3 The Interaction of FXR with Coactivator (A) Comparison of the a and b complexes in the asymmetric unit of the 6ECDCA structure. Helix 12 is shown in yellow, the peptide in the primary coactivator groove is shown in blue, and the second peptide seen in the b complex (of both the 6ECDCA and 3-deoxyCDCA containing structures) is shown in red. The numbers indicate the location of the conserved leucines. (B) A section of a composite omit map (2Fo-Fc) showing electron density for the two coactivator peptides in complex b. The primary coactivator peptide is in blue; the second peptide is in red. (C) A schematic representation showing the contacts of the primary coactivator peptide (blue) with the second peptide (red) and with FXR (residues indicated in boxes). Circles represent hydrogen bonds; dotted lines indicate van der Waals contacts. (D) FXR binding to different NID regions of GRIP1 in the presence of CDCA. The NID-1 peptide has the sequence KGQTKLLQLLTTK, the NID-2 is EKHKILHRLLQDS, and the NID-3 is KENALLRYLLDKD. The NID (1-3) protein consists of the three consecutive NID regions of GRIP-1. (E) Gel filtration chromatography (Pharmacia 100 cm Superdex-75) showing the migration of FXR-LBD, the NID (1-3) region of GRIP-1, and the FXR-LBD + NID (1-3). The elution profiles are shown as a function of increasing time (left to right). The migration of several molecular weight standards is indicated at the top. FXR-LBD elutes at the expected size of a monomer (∼29 kDa). The NID (1-3) polypeptide alone elutes at a larger size (∼46 kDa) than expected (calculated mass ∼24 kDa), presumably due to a noncompact fold. FXR-LBD + NID (1-3) migrates at a size of ∼60 kDa, consistent with one FXR-LBD bound to a more compact NID (1-3) and not consistent with the cooperative binding of two FXR-LBDs subunits with one NID (1-3) fragment (expected migration at a size >82 kDa). Molecular Cell 2003 11, 1093-1100DOI: (10.1016/S1097-2765(03)00112-6) Copyright © 2003 Cell Press Terms and Conditions

Figure 4 Sequences Used for Ligand Binding by the Steroid Receptors Yellow shading shows the position of the α helices; blue shows β strands. Red boxes indicate the residues that contact the steroids as determined from crystal structures (Bledsoe et al., 2002; Brzozowski et al., 1997; Rochel et al., 2000; Sack et al., 2001; Shiau et al., 1998; Williams and Sigler, 1998), and black ovals indicate the residues involved in forming the π-cation molecular switch for ligand-induced activation of FXR. All of the residues in rFXR that contact CDCA-derived ligands are identical with human FXR but not as closely shared with mFXRβ, which has been shown to respond to lanosterol (Otte et al., 2003). The crystal structures of hFXR, mFXRβ, LXRα, LXRβ, and MR are unavailable. Molecular Cell 2003 11, 1093-1100DOI: (10.1016/S1097-2765(03)00112-6) Copyright © 2003 Cell Press Terms and Conditions