Institute of Pharmacy, Faculty of Chemistry and Pharmacy, University of Regensburg, 93040 Regensburg, Germany.
Bioinformatik, Institut für Biochemie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Fahrstraße 17, 91054 Erlangen, Germany.
Int J Mol Sci. 2021 Sep 17;22(18):10047. doi: 10.3390/ijms221810047.
G protein-coupled receptors (GPCRs) are targets of extracellular stimuli and hence occupy a key position in drug discovery. By specific and not yet fully elucidated coupling profiles with α subunits of distinct G protein families, they regulate cellular responses. The histamine H and H receptors (HR and HR) are prominent members of Gs- and Gi-coupled GPCRs. Nevertheless, promiscuous G protein and selective Gi signaling have been reported for the HR and HR, respectively, the molecular mechanism of which remained unclear. Using a combination of cellular experimental assays and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigated the coupling profiles of the HR and HR to engineered mini-G proteins (mG). We obtained coupling profiles of the mGs, mGsi, or mGsq proteins to the HR and HR from the mini-G protein recruitment assays using HEK293T cells. Compared to HR-mGs expressing cells, histamine responses were weaker (pEC, E) for HR-mGsi and -mGsq. By contrast, the HR selectively bound to mGsi. Similarly, in all-atom GaMD simulations, we observed a preferential binding of HR to mGs and HR to mGsi revealed by the structural flexibility and free energy landscapes of the complexes. Although the mG α5 helices were consistently located within the HR binding cavity, alternative binding orientations were detected in the complexes. Due to the specific residue interactions, all mG α5 helices of the HR complexes adopted the Gs-like orientation toward the receptor transmembrane (TM) 6 domain, whereas in HR complexes, only mGsi was in the Gi-like orientation toward TM2, which was in agreement with Gs- and Gi-coupled GPCRs structures resolved by X-ray/cryo-EM. These cellular and molecular insights support (patho)physiological profiles of the histamine receptors, especially the hitherto little studied HR function in the brain, as well as of the pharmacological potential of HR selective drugs.
G 蛋白偶联受体（GPCRs）是细胞外刺激的靶点，因此在药物发现中占据关键地位。通过与不同 G 蛋白家族的α亚基特异性且尚未完全阐明的偶联谱，它们调节细胞反应。组胺 H 和 H 受体（HR 和 HR）是 Gs 和 Gi 偶联 GPCRs 的突出成员。然而，HR 和 HR 分别报道了混杂的 G 蛋白和选择性 Gi 信号转导，其分子机制尚不清楚。我们使用细胞实验测定和高斯加速分子动力学（GaMD）模拟的组合，研究了 HR 和 HR 与工程化 mini-G 蛋白（mG）的偶联谱。我们从使用 HEK293T 细胞的 mini-G 蛋白募集测定中获得了 mGs、mGsi 或 mGsq 蛋白与 HR 和 HR 的偶联谱。与表达 HR-mGs 的细胞相比，组胺反应较弱（pEC，E）用于 HR-mGsi 和 -mGsq。相反，HR 选择性地与 mGsi 结合。同样，在全原子 GaMD 模拟中，我们观察到 HR 优先与 mGs 结合，而 HR 优先与 mGsi 结合，这是通过复合物的结构灵活性和自由能景观揭示的。尽管 mGα5 螺旋始终位于 HR 结合腔内，但在复合物中检测到替代的结合取向。由于特定的残基相互作用，HR 复合物的所有 mGα5 螺旋均采用类似于 Gs 的取向朝向受体跨膜（TM）6 结构域，而在 HR 复合物中，只有 mGsi 采用类似于 Gi 的取向朝向 TM2，这与 X 射线/冷冻电镜解析的 Gs 和 Gi 偶联 GPCRs 结构一致。这些细胞和分子见解支持组胺受体的（病理）生理学特征，特别是迄今为止研究甚少的 HR 在大脑中的功能，以及 HR 选择性药物的药理学潜力。
