Experimental validation of the predicted TGR5 binding mode model

Introduction: TGR5 (Gpbar-1) is a G-protein coupled, membrane-bound bile acid receptor, which is expressed in many human tissues. In the liver TGR5 mediated anti-inflammatory, choleretic and anti-apoptotic effects. Furthermore, the receptor has been linked to bile acid and glucose homeostasis, energy expenditure and inflammatory pathways. Thus, it is conceivable that TGR5 agonists can mediate protective effects in various liver and metabolic diseases. Aim of the present study is the development of a TGR5 structure-function model and a computational and experimental characterization of the ligand binding pocket allow the search for new, specific TGR5 agonists and antagonists.

Methods: 3D structure predictions of the TGR5 receptor were made using homology modelling, furthermore the binding mode of the most potent bile acid ligand, taurolithocholat TLC, was analyzed by molecular docking (Gertzen/Gohlke). This binding mode could be confirmed with a 3D structure-activity relationship analysis (3D-QSAR) with 67 TGR5 ligands (Gertzen/Gohlke). The experimental validation was carried out with the help of TGR5 mutations at positions that show significant interactions with TLC in the predicted binding model. TGR5 variations were introduced into TGR5-cDNA constructs by site-directed mutagenesis. TGR5 function was analyzed through co-transfection of HEK293 cells with TGR5 and a cAMP-sensitive luciferase reporter gene construct. Impact on receptor localization was determined by immunofluorescence microscopy and FACS analysis.

Results: The structure-based model of the TGR5 binding mode, predicted reduced affinity when residues Arg79, Tyr89, Glu169 and Leu244 where mutated, while variation of Tyr240 showed complete loss of function. This was confirmed in mutagenesis studies in transfected HEK293 cells. The model demonstrates no necessary interaction of Ser21 with TLC, which could also be confirmed experimentally. All mutations are correctly localized in the plasma membrane of transfected cells.

Discussion: Mutation of essential residues for TGR5 ligand binding as predicted by a structure-based binding model showed reduced functional activity, which provides strong support for the suggested binding mode of TGR5 agonists. This offers new possibilities to identify not only novel TGR5 agonists but also potential TGR5 antagonists.

Corresponding author: Spomer, Lina

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