Unusual binding of ursodeoxycholic acid to ileal bile acid binding protein: role in activation of FXRα

Abstract

Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its beneficial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We find that UDCA binds to a single site on IBABP and increases the affinity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor α (FXRα) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA.

Fig. 1.

UDCA binds a single site in IBABP. (A) Tryptophan fluorescence spectroscopy was used to quantify the binding affinity of UDCA to IBABP. IBABP (250 µl at 10 µM) was titrated in a stepwise manner with 1 µl increments of UDCA (5 mM). The specific binding measured by normalized changes in emission fluorescence is plotted against UDCA concentration. The hyperbolic binding curve indicates IBABP can bind UDCA in a single binding site. (B, C) The binding sites of UDCA and CA to IBABP were determined by ligand-observed ¹H, ¹⁵N HSQC NMR spectroscopy. The contour plots of NMR spectra of ¹⁵N-GCA (B) or ¹⁵N-GUDCA (C) bound to IBABP at a three-to-one molar ratio are shown. Arrows indicate peaks corresponding to bound bile acids.

Fig. 2.

UDCA increases the binding affinity and lowers the binding cooperativity of IBABP for major human bile acids. Tryptophan fluorescence spectroscopy was used to assess the effect of UDCA on binding affinity and cooperativity of major human bile acids. (A–C) IBABP (270 µl at 20 µM) containing GUDCA (200 µM) was titrated in a stepwise manner with CA, CDCA, or DCA (2.5 mM). The left-shift of the binding curve (closed diamonds) indicates that GUDCA increases the affinity for other bile acids. (D–F) Scatchard plot was used to identify binding cooperativity. ΔF/ΔF_max / [BA] was plotted against ΔF/ΔF_max. It yields a straight line for noncooperative binding, whereas cooperativity results in high curvature of the plot.

Fig. 3.

UDCA induces unique conformational changes in IBABP. Changes to the chemical shift of IBABP upon binding by bile acids were compared using protein-observed ¹H, ¹⁵N HSQC NMR spectra. All complexes are formed by adding bile acids in 10-fold molar excess of [U-¹⁵N]IBABP (100 µM). (A) Overlay of apo-IBABP (black) and TUDCA-bound IBABP (red). (B) Overlay of apo-IBABP (black) and TCDCA-bound IBABP (blue). (C) Overlay of TCDCA-bound IBABP (blue) and TUDCA-bound IBABP (red).

Fig. 4.

UDCA induces strong changes to the chemical shift at bile acid binding site 1. The chemical shifts of residues Gly66, reporting on site 1 (top panels), and Val37, reporting on site 2, (bottom panels) of IBABP were examined in detail in protein-observed ¹H, ¹⁵N HSQC NMR spectra of different bile acid binding. The positions of these residues are shown in apo state (A, E), when bound to TUDCA (B, F), when bound to TCDCA (C, G), and when bound to TUDCA and TCDCA (D, H).

Fig. 5.

UDCA promotes activation of FXRα by bile acids when IBABP is present. Caco-2 cells were transfected with IBABP and treated with UDCA (125 µM) for 5 h. The cells were then treated with CDCA or DCA (25 µM) for 24 h. The relative FXRα activity was determined using a Luciferase reporter assay. Values are the average ± SEM of three independent experiments. ***P < 0.001. The expression levels of the transfected IBABP are shown by Western blot using β-actin as a loading control.

Fig. 6.

IBABP is necessary for the full activation of FXRα by CDCA in Caco-2 cells. Caco-2 cells were treated for 24 h with siRNA targeting IBABP (siIBABP) or scrambled siRNA (siControl). Cells were then treated with UDCA (125 µM) for 5 h followed by incubation with CDCA (25 µM) for 24 h. (A) Activation of FXRα was measured by Luciferase reporter assay. (B) Expression level of the FXRα target gene OSTα was determined by qPCR. Values are the average ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. The knockdown of endogenous IBABP is shown by Western blot using β-actin as a loading control.

Fig. 7.

Model of CDCA bound to IBABP. (A) Model of the binding of two CDCA molecules to human IBABP based on structures of chicken LBABP in complex with two bile acids (1TW4.pdb, 2JU3.pdb). For clarity, we only show the steroid ring A of CDCA at site 1 and ring B of CDCA at site 2. The 7α-OH group of CDCA at site 2 forms a hydrogen bond (dashed line) to the 3α-OH group of the bile acid at site 1. In addition, the ring B 7β-H of CDCA at site 2 interacts with Ile69 in IBABP by Van der Waals forces (arrows). The stereoisomer, UDCA with 7β-OH (gray), cannot form these two interactions. (B) Chemical structure of UDCA, CDCA, CA, and DCA.