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

Abstract

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 H2 and H4 receptors (H2R and H4R) are prominent members of Gs- and Gi-coupled GPCRs. Nevertheless, promiscuous G protein and selective Gi signaling have been reported for the H2R and H4R, 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 H2R and H4R to engineered mini-G proteins (mG). We obtained coupling profiles of the mGs, mGsi, or mGsq proteins to the H2R and H4R from the mini-G protein recruitment assays using HEK293T cells. Compared to H2R–mGs expressing cells, histamine responses were weaker (pEC50, Emax) for H2R–mGsi and –mGsq. By contrast, the H4R selectively bound to mGsi. Similarly, in all-atom GaMD simulations, we observed a preferential binding of H2R to mGs and H4R 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 H2R complexes adopted the Gs-like orientation toward the receptor transmembrane (TM) 6 domain, whereas in H4R 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 H2R function in the brain, as well as of the pharmacological potential of H4R selective drugs.

Molecular Biology of Histamine System, Volume 1

“…Using a novel combination of cellular experimental assays and Gaussian accelerated molecular dynamics (GaMD) simulations, the coupling profiles of the H2R and H4R to engineered mini-G proteins (mG) was explored. Due to the specific residue interactions, all mG α5 helices of the H2R complexes adopted the Gs-like orientation toward the receptor transmembrane (TM) 6 domain, whereas, in H4R complexes, only mGi was in the Gi-like orientation toward TM2, which was in agreement with the respective Gs-and Gi-coupled GPCRs structures resolved by X-ray/cryo-EM [5]. Further studies with the H1R, and in particular the H3R, are needed and, importantly, confirmation of the respective coupling partners in native tissues.…”

Dynamic Insights into the Self-Activation Pathway and Allosteric Regulation of the Orphan G-Protein-Coupled Receptor GPR52

“…In the past few years, the conformational states and structural dynamics of GPCRs have been widely explored by molecular dynamics (MD) simulations. – However, the timescale of the GPCRs’ functional transition is usually not accessible by conventional molecular dynamics (cMD) approaches. Integrated with powerful enhanced sampling techniques, the Gaussian accelerated molecular dynamics (GaMD) simulation has been successfully used to reveal motion details for large proteins such as GPCRs. – In the GaMD algorithm, a harmonic boost potential following the Gaussian distribution is added to smoothen the potential energy surface of the system. It can effectively decrease the energy barrier between different low-free energy basins and thus accelerate conformational sampling. , As an important tool for weak interaction analysis, the independent gradient model (IGM) has been increasingly popular in the visualization of intramolecular and intermolecular interactions in recent years. , In IGM, interaction regions of different strengths are exhibited by isosurfaces of the IGM function with different values.…”