Identification of key amino acid residues in the hTGR5–nomilin interaction and construction of its binding model

TGR5, a member of the G protein-coupled receptor (GPCR) family, is activated by bile acids. Because TGR5 promotes energy expenditure and improves glucose homeostasis, it is recognized as a key target in treating metabolic diseases. We previously showed that nomilin, a citrus limonoid, activates TGR5 and confers anti-obesity and anti-hyperglycemic effects in mice. Information on the TGR5–nomilin interaction regarding molecular structure, however, has not been reported. In the present study, we found that human TGR5 (hTGR5) shows higher nomilin responsiveness than does mouse TGR5 (mTGR5). Using mouse–human chimeric TGR5, we also found that three amino acid residues (Q77ECL1, R80ECL1, and Y893.29) are important in the hTGR5–nomilin interaction. Based on these results, an hTGR5–nomilin binding model was constructed using in silico docking simulation, demonstrating that four hydrophilic hydrogen-bonding interactions occur between nomilin and hTGR5. The binding mode of hTGR5–nomilin is vastly different from those of other TGR5 agonists previously reported, suggesting that TGR5 forms various binding patterns depending on the type of agonist. Our study promotes a better understanding of the structure of TGR5, and it may be useful in developing and screening new TGR5 agonists.TGR5作为G蛋白偶联受体（G protein-coupled receptor, GPCR）家族成员，可被胆汁酸激活。鉴于TGR5能够促进能量消耗、改善葡萄糖稳态，其被视为治疗代谢疾病的关键靶点。既往研究表明，柑橘柠檬苦素类化合物诺米林（nomilin）可激活TGR5，并在小鼠体内发挥抗肥胖与抗高血糖功效。然而，目前尚未有关于TGR5与诺米林相互作用的分子结构相关报道。本研究发现，人源TGR5（hTGR5）对诺米林的响应性高于小鼠TGR5（mTGR5）。通过构建人鼠嵌合TGR5，我们进一步确认，三个氨基酸残基（Q77ECL1、R80ECL1及Y893.29）在hTGR5与诺米林的相互作用中发挥关键作用。基于上述结果，我们通过虚拟（in silico）对接模拟构建了hTGR5与诺米林的结合模型，结果显示诺米林与hTGR5之间存在四处亲水性氢键相互作用。hTGR5与诺米林的结合模式与此前报道的其他TGR5激动剂存在显著差异，这提示TGR5可根据激动剂类型形成多样化的结合模式。本研究有助于加深对TGR5结构的理解，可为新型TGR5激动剂的开发与筛选提供参考依据。