Specific Engineered G Protein Coupling to Histamine Receptors Revealed from Cellular Assay Experiments and Accelerated Molecular Dynamics Simulations

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(2) and H(4) receptors (H(2)R and H(4)R) are prominent members of Gs- and Gi-coupled GPCRs. Nevertheless, promiscuous G protein and selective Gi signaling have been reported for the H(2)R and H(4)R, 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 H(2)R and H(4)R to engineered mini-G proteins (mG). We obtained coupling profiles of the mGs, mGsi, or mGsq proteins to the H(2)R and H(4)R from the mini-G protein recruitment assays using HEK293T cells. Compared to H(2)R–mGs expressing cells, histamine responses were weaker (pEC(50), E(max)) for H(2)R–mGsi and –mGsq. By contrast, the H(4)R selectively bound to mGsi. Similarly, in all-atom GaMD simulations, we observed a preferential binding of H(2)R to mGs and H(4)R 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 H(2)R complexes adopted the Gs-like orientation toward the receptor transmembrane (TM) 6 domain, whereas in H(4)R 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 H(2)R function in the brain, as well as of the pharmacological potential of H(4)R selective drugs.