Castel Jérôme, Botzanowski Thomas, Brooks Ieva, Frechard Alexandre, Bey Gilbert, Schroeter Marine, Del Nero Elise, Debaene François, Ciesielski Fabrice, Zeyer Denis, Cianferani Sarah, Morales Renaud
Department of Biophysics, Novalix, 16 Rue d'Ankara, 67000, Strasbourg, France.
Laboratoire de Spectrométrie de Masse BioOrganique, IPHC UMR 7178, Infrastructure Nationale de Protéomique ProFI, Université de Strasbourg, CNRS, UAR2048 CNRS, 67087, Strasbourg, France.
Sci Rep. 2025 Aug 26;15(1):31517. doi: 10.1038/s41598-025-16529-w.
Many physiological processes are dependent on G protein-coupled receptors (GPCRs), the biggest family of human membrane proteins and a significant class of therapeutic targets. Once activated by external stimuli, GPCRs use G proteins and arrestins as transducers to generate second messengers and trigger downstream signaling, leading to diverse signaling profiles. The G protein-coupled bile acid receptor 1 (GPBAR1, also known as Takeda G protein-coupled receptor 5, TGR5) is a class A bile acid membrane receptor that regulates energy homeostasis and glucose and lipid metabolism. GPBAR1/Gs protein interactions are implicated in the prevention of diabetes and the reduction of inflammatory responses, making GPBAR1 a potential therapeutic target for metabolic disorders. Here, we present combined hydrogen/deuterium exchange mass spectrometry (HDX-MS) and cryo-electron microscopy (cryo-EM) to identify the molecular determinants of GPBAR1 conformational dynamics upon G protein binding. Thanks to extensive optimization, we achieved over 75% sequence coverage by HDX-MS of a complete GPCR complex and a 2.5 Å resolution structure by cryo-EM, both of which are state-of-the-art. Altogether, our results provide information on the under-investigated GPBAR1 binding mode to its cognate G protein, pinpointing the synergic and powerful combination of higher cryo-EM and lower HDX-MS resolution techniques to dissect GPCR/G protein binding characteristics.
许多生理过程都依赖于G蛋白偶联受体（GPCRs），它是人类膜蛋白中最大的家族，也是一类重要的治疗靶点。一旦被外部刺激激活，GPCRs就会利用G蛋白和抑制蛋白作为转导分子来产生第二信使并触发下游信号传导，从而导致多种信号传导模式。G蛋白偶联胆汁酸受体1（GPBAR1，也称为武田G蛋白偶联受体5，TGR5）是一种A类胆汁酸膜受体，可调节能量稳态以及葡萄糖和脂质代谢。GPBAR1与Gs蛋白的相互作用与预防糖尿病和减轻炎症反应有关，这使得GPBAR1成为代谢紊乱的潜在治疗靶点。在此，我们采用氢/氘交换质谱联用（HDX-MS）和冷冻电子显微镜（cryo-EM）技术来确定G蛋白结合后GPBAR1构象动力学的分子决定因素。经过广泛优化，我们通过HDX-MS实现了对完整GPCR复合物超过75%的序列覆盖，并通过cryo-EM获得了分辨率为2.5 Å的结构，这两项成果均处于当前技术水平。总之，我们的研究结果提供了关于研究较少的GPBAR1与其同源G蛋白结合模式的信息，明确了更高分辨率的cryo-EM和较低分辨率的HDX-MS技术相结合在剖析GPCR/G蛋白结合特性方面的协同作用和强大功能。
