Conformational dynamics of human FXR-LBD ligand interactions studied by hydrogen/deuterium exchange mass spectrometry: insights into the antagonism of the hypolipidemic agent Z-guggulsterone

Abstract

Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of transcription factors that plays a key role in the regulation of bile acids, lipid and glucose metabolisms. The regulative function of FXR is governed by conformational changes of the ligand binding domain (LBD) upon ligand binding. Although FXR is a highly researched potential therapeutic target, only a limited number of FXR-agonist complexes have been successfully crystallized and subsequently yielded high resolution structures. There is currently no structural information of any FXR-antagonist complexes publically available. We therefore explored the use of amide hydrogen/deuterium exchange (HDX) coupled with mass spectrometry for characterizing conformational changes in the FXR-LBD upon ligand binding. Ligand-specific deuterium incorporation profiles were obtained for three FXR ligand chemotypes: GW4064, a synthetic non-steroidal high affinity agonist; the bile acid chenodeoxycholic acid (CDCA), the endogenous low affinity agonist of FXR; and Z-guggulsterone (GG), an in vitro antagonist of the steroid chemotype. A comparison of the HDX profiles of their ligand-bound FXR-LBD complexes revealed a unique mode of interaction for GG. The conformational features of the FXR-LBD-antagonist interaction are discussed.

Keywords: Conformational dynamics; Farnesoid X receptor; Guggulsterone; Hydrogen/deuterium exchange; Ligand interaction; Mass spectrometry.

Figure 1

Fluorescence spectra of FXR-LBD with structures of the ligands used in the current study: GW4064, CDCA, and GG (Z)

Figure 2

Deuterium levels observed for the FXR-LBD with and without ligands after the following incubation periods: 0.5, 1, 2, 5, 10, 30, 60min.

Figure 3

Comparative presentation of the HDX heat maps of FXR-LBD in the presence of ligands. Each horizontal color block represents an analyzed peptic peptide, and each block contains seven time points (from top: 0.5, 1, 2, 5, 10, 30, 60 min).

Figure 4

Exchange-in plots of peptides that showed disparate exchange-in characteristics. (A) Exchange-in kinetics: apo-FXR-LBD, red line; GW4064, blue line; CDCA, green line; GG, yellow line. (B) X-ray structure of FXR-LBD (green) bound to GW4064 (orange) (PDB ID 3DCT); peptides that showed disparate exchange-in characteristics in presence of the diverse ligands are colored blue.

Figure 4

Exchange-in plots of peptides that showed disparate exchange-in characteristics. (A) Exchange-in kinetics: apo-FXR-LBD, red line; GW4064, blue line; CDCA, green line; GG, yellow line. (B) X-ray structure of FXR-LBD (green) bound to GW4064 (orange) (PDB ID 3DCT); peptides that showed disparate exchange-in characteristics in presence of the diverse ligands are colored blue.

Figure 5

Average differences in deuterium levels (in %) of FXR-LBD with or without ligand overlaid onto crystallographic structures. The N-terminal region which is covered by the peptides 201–222 and 229–241 is not visible in the crystal structures. Top row: Co-crystal structure of FXR-LBD ligand complex, PDB ID 3DCT for (A) and PDB ID 1OSV for (B) and (C). Contact residues between ligand and protein are colored in pink. The ligand itself is colored in orange. Bottom row: Average differences in deuterium levels (D%) of seven time points (0.5, 1, 2, 5, 10, 30, and 60 min) are mapped onto the crystal structure of FXR-LBD-ligand complex, PDB ID 3DCT for (D) and PDB ID 1OSV for (E) and (F). Regions that were not covered by the current experiments are depicted in grey. NS, “not significant”.

Figure 6

Differential HDX data of FXR-LBD-agonist and FXR-LBD-antagonist. Left: Deuterium level differences in percentage (D %) from one complex to another are shown at different time points (0.5, 1, 2, 5, 10, 30, and 60 min, from left to right) for each peptide. Right: Average differences in deuterium percentage (D %) of seven time points (0.5, 1, 2, 5, 10, 30, and 60 min) were overlaid onto the crystallographic structure, PDB ID 3DCT for (A) and (B), PDB ID 1OSV for (C). Positive numbers indicate higher deuterium levels and thus less protection in the FXR-LBD-GG relative to the FXR-LBD-GW4064. Negative numbers indicate lower deuterium levels and thus more protection in the FXR-LBD-GG relative to the FXR-LBD-GW4064.