In a study published online in Nature on July 22, Prof. ZHANG Yan  from Zhejiang University, School of Medicine, Prof. XIE Xin from the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences, and Profs. YU Xiao and SUN Jinpeng from Shandong University, determined for the first time the 3Å cryo-EM structures of the GPBAR–Gs in complexes with P395 and INT-777, a highly potent synthetic agonist and a semisynthesized bile acid derivative. Moreover, it is launching the integrated systematic approaches to navigate precision medicine in Zhejiang University Medical Center (Liangzhu Lab).

Playing a central role in the liver-bile acids-microbiota axis, bile acids and their receptors are the research hot spot in biomedical fields. Studies have demonstrated that bile acids and their derivatives might be used to treat diseases like primary biliary cholangitis (PBC), non-alcoholic steatohepatitis (NASH), type 2 diabetes. Bile acids exert regulatory functions by activating the membrane receptor GPBAR/TGR5 and the nuclear receptor FXR. GPBAR belongs to the G protein-coupled receptor (GPCR) superfamily. Many of the beneficial effects of bile acids have been attributed to GPBAR–Gs coupling. In addition, GPBAR signals to β-arrestin to induce innate antiviral immune response.  Understanding at molecular level that how GPBAR recognizes so many different bile acids and how to induce downstream signaling bias will greatly facilitate our understanding of this receptor, and promote future drug discovery.  

With the structural information and the biochemical assays, scientists revealed how GPBAR recognize amphipathic molecules. GPBAR expands a large ovate pocket to accommodate the bulky bile acids. The TM5, ECL2 and ECL3, form a hydrophobic wall which interact with the hydrophobic core of bile acids, while the hydrophilic side of bile acids form specific interactions with hydrophilic amino acids on TM6 and TM7.

Meanwhile, they elucidated how amino acid finger prints recognizing various bile acids. Endogenous bile acids have the same core but are differentiated mainly by hydroxylation at various position. By analyzing the binding mode of INT-777 with GPBAR, the scientists reveal that triplet leucine cluster (L244^6.55, L263^7.36 and L266^7.39), as well as a potential role of S247^6.58, constitute a fingerprint reader to discriminate the interactions between different bile acids and GPBAR.

Besides, the team discovered a second binding pockets for allosteric bile acid binding. Allosteric binding pockets are commonly discovered in GPCRs, but an allosteric binding pocket for native ligand is very unusual. Scientists discovered that the intracellular side of TM3-5 of GPBAR form a second bile acid binding pocket, many bile acids with 12-OH structure could bind to this pocket and regulate receptor activation with ligand in the orthosteric pocket. This may explain the diverse functions of diverse bile acids, and provide novel insight to future drug design.

“We elucidated a non-classical activation mechanism of GPBAR and the previous unclear function of ICL3 of GPCRs,” said ZHANG Yan, a research at the Zhejiang University School of Medicine, and a senior author of the study. “and demonstrated the structural basis of biased signaling elicited by different ligands.”

This study not only revealed the structural basis of GPBAR-bile acids recognition and signaling bias, but also discovered a unique activation mechanism which is different from all GPCRs with known structures.Considering the diverse and the important roles of GPBAR and bile acids in regulating metabolic and immune responses, the high-resolution structures of GPBAR-ligand-Gs complexes may greatly facilitate rational drug design and discovery targeting GPBAR.

Link to the article: https://www.nature.com/articles/s41586-020-2569-1