Impaired transitioning from an inactive to an active state of FXR underlies a PFIC5 phenotype

Nuclear receptor FXR acts as a key regulator within bile homeostasis and metabolism. Within the enterohepatic cycle, reabsorbed bile acids act as agonists on FXR, which transcriptionally controls the synthesis and transport of bile acids. Binding occurs in the ligand-binding domain (LBD), favoring a conformational change to the active state in which Helix 12 interacts with the LBD to form an interaction surface for nuclear co-activators. The homozygous missense variant p.(Thr296Ile) (NM_001206979.2: c.887C>T in the FXR-encoding NR1H4 gene), identified in a PFIC5 patient (Pfister et al., 2022), is located close to this critical conformational change. The variant protein showed reduced transcriptional activity as measured via Luciferase assay on the downstream targets BSEP and SHP. Immunofluorescence staining and Western Blot assay showed normal protein localization and expression levels, indicating that the decreased transcriptional activity is indeed coupled to decreased protein activity. Using molecular dynamics simulations, we analyzed the dynamics of the conformational change from an inactive to an active state of the FXR LBD. While the wildtype protein frequently changes into the active state, this movement and the necessary perfect placement of Helix 12 was significantly impeded within the variant protein. Overall, this is the first study to sample the conformational change from an inactive to an active state within the FXR LBD and, thus, might enable critical insights into specifically targeting FXR activity. Our results, a comprehensive combination of in vitro and in silico experiments, reveal in depth the molecular mechanism of a patient-associated missense variant.

Publication History

Article published online:
23 January 2024