Isolation and conformational analysis of the Gα α-helical domain.

Heterotrimeric G proteins (G proteins), composed of Gα, Gß, and Gγ subunits, are the major downstream signaling molecules of the G protein-coupled receptors. Upon activation, Gα undergoes conformational changes both in the Ras-like domain (RD) and the α-helical domain (AHD), leading to the dissociation of Gα from Gßγ and subsequent regulation of downstream effector proteins. Gα RD mediate the most of classical functions of Gα. However, the role of Gα AHD is relatively not well elucidated despite its much higher sequence differences between Gα subtypes than those between Gα RD. Here, we isolated AHD from Gαs, Gαi1, and Gαq to provide tools for examining Gα AHD. We investigated the conformational dynamics of the isolated Gα AHD compared to those of the GDP-bound Gα. The results showed higher local conformational dynamics of Gα AHD not only at the domain interfaces but also in regions further away from the domain interfaces. This finding is consistent with the conformation of Gα AHD in the receptor-bound nucleotide-free state. Therefore, the isolated Gα AHD could provide a platform for studying the functions of Gα AHD, such as identification of the Gα AHD-binding proteins.

Gαs slow conformational transition upon GTP binding and a novel Gαs regulator.

G proteins are major signaling partners for G protein-coupled receptors (GPCRs). Although stepwise structural changes during GPCR-G protein complex formation and guanosine diphosphate (GDP) release have been reported, no information is available with regard to guanosine triphosphate (GTP) binding. Here, we used a novel Bayesian integrative modeling framework that combines data from hydrogen-deuterium exchange mass spectrometry, tryptophan-induced fluorescence quenching, and metadynamics simulations to derive a kinetic model and atomic-level characterization of stepwise conformational changes incurred by the ß2-adrenergic receptor (ß2AR)-Gs complex after GDP release and GTP binding. Our data suggest rapid GTP binding and GTP-induced dissociation of Gαs from ß2AR and Gßγ, as opposed to a slow closing of the Gαs α-helical domain (AHD). Yeast-two-hybrid screening using Gαs AHD as bait identified melanoma-associated antigen D2 (MAGE D2) as a novel AHD-binding protein, which was also shown to accelerate the GTP-induced closing of the Gαs AHD.

The Conformational Dynamics of Heterotrimeric G Proteins During GPCR-Mediated Activation.

Heterotrimeric G proteins (G proteins) are essential cellular signaling proteins that mediate extracellular signals to achieve various cellular functions. G-protein-coupled receptors (GPCRs) are the major guanine nucleotide exchange factors (GEFs) that induce G proteins to release guanosine diphosphate and rapidly bind to guanosine triphosphate, resulting in G protein activation. G proteins undergo dynamic conformational changes during the activation/inactivation process, and the precise structural mechanism of GPCR-mediated G protein activation is of great interest. Over the last decade, a number of GPCR-G protein complex structures have been identified, yet an understanding of the mechanisms underlying allosteric conformational changes during receptor-mediated G protein activation and GPCR-G protein coupling selectivity is only now emerging. This review discusses recent studies on the dynamic conformational changes of G proteins and provides insight into the structural mechanism of GPCR-mediated G protein activation.

Effect of α-helical domain of Gi/o α subunit on GDP/GTP turnover.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of α, ß, and γ subunits, and Gα has a GDP/GTP-binding pocket. When a guanine nucleotide exchange factor (GEF) interacts with Gα, GDP is released, and GTP interacts to Gα. The GTP-bound activated Gα dissociates from GEF and Gßγ, mediating the induction of various intracellular signaling pathways. Depending on the sequence similarity and cellular function, Gα subunits are subcategorized into four subfamilies: Gαi/o, Gαs, Gαq/11, and Gα12/13. Although the Gαi/o subtype family proteins, Gαi3 and GαoA, share similar sequences and functions, they differ in their GDP/GTP turnover profiles, with GαoA possessing faster rates than Gαi3. The structural factors responsible for these differences remain unknown. In this study, we employed hydrogen/deuterium exchange mass spectrometry and mutational studies to investigate the factors responsible for these functional differences. The Gα subunit consists of a Ras-like domain (RD) and an α-helical domain (AHD). The RD has GTPase activity and receptor-binding and effector-binding regions; however, the function of the AHD has not yet been extensively studied. In this study, the chimeric construct containing the RD of Gαi3 and the AHD of GαoA showed a GDP/GTP turnover profile similar to that of GαoA, suggesting that the AHD is the major regulator of the GDP/GTP turnover profile. Additionally, site-directed mutagenesis revealed the importance of the N-terminal part of αA and αA/αB loops in the AHD for the GDP/GTP exchange. These results suggest that the AHD regulates the nucleotide exchange rate within the Gα subfamily.

The conformational transition during G protein-coupled receptor (GPCR) and G protein interaction.

The precise structural mechanism of G protein-coupled receptor (GPCR)-G protein coupling has been of significant research interest because it provides fundamental knowledge on cellular signaling and valuable information for GPCR-targeted drug development. Over the last decade, several GPCR-G protein complex structures have been identified. However, these structures are mere snapshots of guanosine diphosphate (GDP)-released stable GPCR-G protein complexes, which have limited the understanding of the allosteric conformational transition during receptor binding to GDP release and the GPCR-G protein coupling selectivity. Recently, deeper insights into the mechanism underlying stepwise conformational changes during GPCR-G protein coupling were obtained using hydrogen/deuterium exchange mass spectrometry, hydroxyl radical footprinting-mass spectrometry, X-ray crystallography, cryoelectron microscopy, and molecular dynamics simulation techniques. This review summarizes these recent developments.

Conformational switch that induces GDP release from Gi.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of α, ß, and γ subunits. Gα switches between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active states, and Gßγ interacts with the GDP-bound state. The GDP-binding regions are composed of two sites: the phosphate-binding and guanine-binding regions. The turnover of GDP and GTP is induced by guanine nucleotide-exchange factors (GEFs), including G protein-coupled receptors (GPCRs), Ric8A, and GIV/Girdin. However, the key structural factors for stabilizing the GDP-bound state of G proteins and the direct structural event for GDP release remain unclear. In this study, we investigated structural factors affecting GDP release by introducing point mutations in selected, conserved residues in Gαi3. We examined the effects of these mutations on the GDP/GTP turnover rate and the overall conformation of Gαi3 as well as the binding free energy between Gαi3 and GDP. We found that dynamic changes in the phosphate-binding regions are an immediate factor for the release of GDP.

Structural mechanism underlying primary and secondary coupling between GPCRs and the Gi/o family.

Heterotrimeric G proteins are categorized into four main families based on their function and sequence, Gs, Gi/o, Gq/11, and G12/13. One receptor can couple to more than one G protein subtype, and the coupling efficiency varies depending on the GPCR-G protein pair. However, the precise mechanism underlying different coupling efficiencies is unknown. Here, we study the structural mechanism underlying primary and secondary Gi/o coupling, using the muscarinic acetylcholine receptor type 2 (M2R) as the primary Gi/o-coupling receptor and the ß2-adrenergic receptor (ß2AR, which primarily couples to Gs) as the secondary Gi/o-coupling receptor. Hydrogen/deuterium exchange mass spectrometry and mutagenesis studies reveal that the engagement of the distal C-terminus of Gαi/o with the receptor differentiates primary and secondary Gi/o couplings. This study suggests that the conserved hydrophobic residue within the intracellular loop 2 of the receptor (residue 34.51) is not critical for primary Gi/o-coupling; however, it might be important for secondary Gi/o-coupling.