Salze Marine, Chrétien Sébastien, Boora Tegvir S, Macovei Madalina, Barbeau Eric, Blais Véronique, Laporte Stéphane A, Audet Martin
Department of Pharmacology and Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada.
Division of Clinical and Translational Research, Department of Medicine, McGill University, Montréal, Québec, Canada.
PLoS One. 2025 Jul 18;20(7):e0320114. doi: 10.1371/journal.pone.0320114. eCollection 2025.
Prostaglandin F2ɑ (PGF2ɑ) is a bioactive lipid derived from arachidonic acid and is involved in many physiological and pathophysiological processes, such as parturition, vascular tone regulation, glaucoma and inflammation. It acts by binding to the Prostaglandin F2ɑ receptor (FP), a G Protein-Coupled Receptor (GPCR) that mediates signaling events by engaging intracellular heterotrimeric G protein effectors. The orthosteric binding site of lipid-binding receptors displays greater efficacy-dependent plasticity that hinders the design of ligands. Solving the structure of FP with ligands of different efficacies at an atomic level is important to fully understand its mechanism of activation and inhibition. Most purified FP-ligand complexes are unstable in vitro. The development of new X-ray crystallography and single particle cryo-electron microscopy (cryoEM) strategies to understand receptors' signal transduction requires improved purification yield and in vitro stability of the receptor. Here, we present a protein engineering effort to optimize the FP protein sequence for use in structural biology. Strategies involve protein insertion sites in the third intracellular loop (ICL3), N-terminal and C-terminal deletions, and single-point mutations that favorably affect receptor purification yield and stability in vitro. The best FP construct displays a yield of 1.5 mg/L and a stability of 59oC, which constitute a threefold improvement in purification yield and 9oC increase in stability over the wild-type receptor. These modifications in the receptor are suitable for pursuing alternative strategies for improving FP purification yield and for studying FP binding efficacy to its ligands through structural biology approaches.
前列腺素F2α（PGF2α）是一种源自花生四烯酸的生物活性脂质，参与许多生理和病理生理过程，如分娩、血管张力调节、青光眼和炎症。它通过与前列腺素F2α受体（FP）结合发挥作用，FP是一种G蛋白偶联受体（GPCR），通过与细胞内异源三聚体G蛋白效应器结合介导信号转导事件。脂质结合受体的正构结合位点表现出更大的效能依赖性可塑性，这阻碍了配体的设计。在原子水平上解析具有不同效能的配体与FP的结构对于全面理解其激活和抑制机制至关重要。大多数纯化的FP-配体复合物在体外不稳定。开发新的X射线晶体学和单颗粒冷冻电子显微镜（cryoEM）策略以了解受体的信号转导需要提高受体的纯化产量和体外稳定性。在这里，我们进行了一项蛋白质工程工作，以优化用于结构生物学的FP蛋白序列。策略包括在第三细胞内环（ICL3）中的蛋白质插入位点、N端和C端缺失以及单点突变，这些对受体的纯化产量和体外稳定性有有利影响。最佳的FP构建体产量为1.5 mg/L，稳定性为59℃，与野生型受体相比，纯化产量提高了三倍，稳定性提高了9℃。受体的这些修饰适用于寻求提高FP纯化产量的替代策略，以及通过结构生物学方法研究FP与其配体的结合效能。
