Fig. 4.

¹H/¹⁵N-HSQC spectra of fully labeled FXR-LBD. Left panels show superposition of apo and apo+ligand, right panels show superposition of apo and apo + ligand + co-activator peptide. Blue—apo; red—with ligand; cyan—with ligand and co-activator peptide (NCoA). Concentrations of protein (200 µM), peptide (500 µM) and ligand (500 µM) were constant for all experiments to ensure saturation. All spectra were processed and compared at the same S/N. A representative signal is shown in each panel to visualise equal S/N. In unliganded state, the ¹H/¹⁵N-HSQC reveals only a fraction of all signals indicating conformational flexibility of part of the FXR-LBD. Upon addition of an FXR agonist (CDCA, GW4064 or fexaramine), additional but still not all signals appear suggesting partial stabilisation of the protein. Addition of co-activator peptide causes marked further stabilisation as observed by appearance of almost all ¹H/¹⁵N-HSQC signals. Addition of the FXR partial agonists ivermectin or 1 also induced stabilisation of the FXR-LBD but in contrast to the FXR agonists, addition of co-activator peptide had no further effect. Addition of the FXR antagonist guggulsterone had only minor/no effect on the HSQC indicating much less stabilisation of the FXR-LBD