Heterotrimeric G proteins are key intracellular coordinators that receive signals from cells through activation of cognate G protein-coupled receptors (GPCRs). The details of their atomic interactions and structural mechanisms have been described by many biochemical and biophysical studies. Specifically, a framework for understanding conformational changes in the receptor upon ligand binding and associated G protein activation was provided by description of the crystal structure of the β2-adrenoceptor-Gs complex in 2011. This review focused on recent findings in the conformational dynamics of G proteins and GPCRs during activation processes.
Heterotrimeric guanine nucleotide-binding proteins (G proteins) are signal transducers that play a crucial role in mediating downstream signal transduction of G protein-coupled receptors (GPCRs) (Ross and Gilman, 1980). G proteins contain three subunits, α, β, and γ. In their inactive state, the Gα subunit binds to guanosine diphosphate (GDP) to form a stable complex with Gβγ partners. When agonist-activated GPCRs couple to the GDP-bound form of G proteins, GDP is replaced by guanosine triphosphate (GTP), which induces dissociation of the Gα subunit from GPCR and Gβγ subunits (Fig. 1A). The GTP-bound Gα subunit or the Gβγ subunits transduce signals through interaction with downstream effectors (Hamm, 1998). Finally, the signal is terminated by the intrinsic GTPase catalytic activity of the Gα subunit, which hydrolyzes GTP to GDP, enabling recruitment of Gβγ subunits to form inactive heterotrimers (Fig. 1A).
There are 21 known isoforms encoded by 16 Gα subunit genes, 6 documented Gβ subunits encoded by 5 genes, and 12 reported Gγ subunits in human (Simon
The Gs family includes 2 isoforms, Gs and Golf, which signal via stimulation of second messengers such as cAMP, as well as Src tyrosine kinase and protein kinase A (Neves
In the 1990s, the structures of Gα subunits in GTP- and GDP-bound forms were described as either a monomer or a Gαβγ heterotrimer (Noel
Various biochemical and biophysical studies have investigated GPCR-mediated G protein activation (Preininger
The distance between GPCR-G protein contact sites and the nucleotide-binding pocket is approximately 30 Å (Fig. 2A), and therefore an allosterical regulation induced by GPCRs should be existed to transform signal from the binding sites to the nucleotide-binding pocket to trigger the release of GDP from Gα subunit. A number of recent studies sought to define the allosteric conformational changes in G proteins upon GPCR binding using
The C-terminus of the Gα subunit is the major GPCR contact site (Fig. 2B), and therefore the interaction between a GPCR and the C-terminus of the Gα subunit may induce conformational changes allosterically in Gα through the α5 helix. Recent modeling and experimental studies predicted the critical role of the α5 helix in G protein activation by GPCRs. Using a combination of mutagenesis and MD simulation, Shim and colleagues first described the molecular basis of cannabinoid CB1 receptor coupling to heterotrimeric Gαiβγ proteins (Shim
The displacement or rotation of the α5 helix appears to be linked to the perturbation of intramolecular interactions in the Gα subunit, which would facilitate GDP release (Fig. 3A). Dror
A comprehensive analysis of available Gα crystal structures further emphasized the role of the α5 helix as a bridge for GPCR-mediated allosteric GDP release and suggested the α1 helix as a “hub”; the α1 helix links various important functional regions of Gα including the N-terminus of the α5 helix, AH domain, and GDP through universally conserved residues (Flock
The hydrophobic region surrounded by the αN/β1 hinge and the β2/β3 loop of Gαs is another major contact site with receptors (Fig. 3A), and the interaction of receptors with this region may induce allosteric conformational changes at the nucleotide-binding pocket through the β1 strand. A crosslinking study together with MD simulation data indicated the high conservation of Phe139 in ICL2 of cannabinoid CB2 receptor anchors in a hydrophobic triad formed by residues from the αN/β1 hinge, β2/β3 loop, and α5 helix of Gαi1 (Mnpotra
When GDP or GTP is bound to G proteins, Ras and AH domains are in the “close state” based on the X-ray crystal structures (Fig. 1). The interface between the two domains is comprised of interactions between the α1, αA, and αF helices and the linker 1 (α1/αA loop) and between the αG and αA helices, the β4/α3 loop, the αD/αE loop, and the switch 1 (Fig. 1D). It is noteworthy that the residues responsible for the interdomain interactions are highly conserved in all Gα family proteins (Flock
Taken together, these combined studies show structural dynamics and conformational relevance of distinct states containing GDP, GTP, and receptor-bound Gα, allowing us to gain insight into the activation process of G proteins which start from the GDP-bound form and progress to the nucleotide-free state or receptor bound form and finally to the GTP bound form or active state (Fig. 1A). The activation mechanism suggests the involvement of receptor induced allosteric conformational changes in the Gα subunit through two major interactive sites, which was clearly identified previously (Duc
It has been known that agonist-bound receptors adopt multiple conformations equilibrated between inactive and active states (Nygaard
The modeling study suggests the existence of an inverse correlation between the ligand binding site and the G protein binding interface (Kolan
Together, these data suggest that coupling to G protein and subsequent nucleotide release is sufficient to promote stabilization of the active state of the receptor or “a closed receptor conformation”, preventing ligand access to and/or exit from the orthosteric ligand-binding site. Despite structural variance, the stabilization of G proteins in structural changes of GPCRs might be shared throughout GCPRs. Similar findings were observed in several families of GPCRs including the muscarinic receptor, the opioid receptor, and the ghrelin receptor (Mary
The high-resolution crystal structure of β2AR-Gs provides an excellent model to carry out a large number of computational and biochemical/biophysical studies in order to understand the conformational mechanism of G protein activation. Combined with previous findings over the last thirty years, these studies provide us with more details about the structural mechanism of the G protein activation cycle. However, there are still more questions to be answered to develop a concrete model for the G protein activation processes.
Although many structural and functional assays have been used to indicate several critical regions in either the Gα subunit or GPCR that are responsible for selectivity, understanding how various ligand-induced conformational changes in GPCRs allow recognition of specific cognate G proteins still remains challenging. Several studies have reported models of GPCR-G protein complexes by using β2AR-Gs structures as a model (Shim
Another question yet to be answered is the conformational sequence of G protein activation by GPCRs. Two major regions in the Gα subunit are involved in the GPCR interaction: the C-terminus of the α5 helix and αN/β1 hinge as described in this review. However, we still do not have definitive answers on the conformational steps of GPCR-G protein activation or which regions have a major role in the initial release of GDP.
Besides G proteins, arrestins also have important roles in GPCR signaling in relation to G protein-independent signal transduction. Recently, great achievements have been made to understand the structural mechanism of the GPCR-mediated arrestin activation process (Park
This work was supported by the National Research Foundation of Korea funded by the Korean government (NFR-2015R1A1A1A05027473 and NRF-2012R1A5A2A28671860).