Gα C-terminus Research Articles

Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to G\u03b1i1

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the nucleotide-free state of Gα.

Ric-8A, a GEF, and a Chaperone for G Protein α-Subunits: Evidence for the Two-Faced Interface.

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a prominent non-receptor GEF and a chaperone of G protein α-subunits (Gα). Recent studies shed light on the structure of Ric-8A, providing insights into the mechanisms underlying its interaction with Gα. Ric-8A is composed of a core armadillo-like domain and a flexible C-terminal tail. Interaction of a conserved concave surface of its core domain with the Gα C-terminus appears to mediate formation of the initial Ric-8A/GαGDP intermediate, followed by the formation of a stable nucleotide-free complex. The latter event involves a large-scale dislocation of the Gα α5-helix that produces an extensive primary interface and disrupts the nucleotide-binding site of Gα. The distal portion of the C-terminal tail of Ric-8A forms a smaller secondary interface, which ostensibly binds the switch II region of Gα, facilitating binding of GTP. The two-site Gα interface of Ric-8A is distinct from that of GPCRs, and might have evolved to support the chaperone function of Ric-8A.

Structural underpinnings of Ric8A function as a G-protein \u03b1-subunit chaperone and guanine-nucleotide exchange factor

Resistance to inhibitors of cholinesterase 8A (Ric8A) is an essential regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor and a chaperone. We report two crystal structures of Ric8A, one in the apo form and the other in complex with a tagged C-terminal fragment of Gα. These structures reveal two principal domains of Ric8A: an armadillo-fold core and a flexible C-terminal tail. Additionally, they show that the Gα C-terminus binds to a highly-conserved patch on the concave surface of the Ric8A armadillo-domain, with selectivity determinants residing in the Gα sequence. Biochemical analysis shows that the Ric8A C-terminal tail is critical for its stability and function. A model of the Ric8A/Gα complex derived from crosslinking mass spectrometry and molecular dynamics simulations suggests that the Ric8A C-terminal tail helps organize the GTP-binding site of Gα. This study lays the groundwork for understanding Ric8A function at the molecular level.

Ric-8A, a G protein chaperone with nucleotide exchange activity induces long-range secondary structure changes in Gα.

Cytosolic Ric-8A has guanine nucleotide exchange factor (GEF) activity and is a chaperone for several classes of heterotrimeric G protein α subunits in vertebrates. Using Hydrogen-Deuterium Exchange-Mass Spectrometry (HDX-MS) we show that Ric-8A disrupts the secondary structure of the Gα Ras-like domain that girds the guanine nucleotide-binding site, and destabilizes the interface between the Gαi1 Ras and helical domains, allowing domain separation and nucleotide release. These changes are largely reversed upon binding GTP and dissociation of Ric-8A. HDX-MS identifies a potential Gα interaction site in Ric-8A. Alanine scanning reveals residues crucial for GEF activity within that sequence. HDX confirms that, like G protein-coupled receptors (GPCRs), Ric-8A binds the C-terminus of Gα. In contrast to GPCRs, Ric-8A interacts with Switches I and II of Gα and possibly at the Gα domain interface. These extensive interactions provide both allosteric and direct catalysis of GDP unbinding and release and GTP binding.

Evidence that the Rhodopsin Kinase (GRK1) N-Terminus and the Transducin Gα C-Terminus Interact with the Same "Hydrophobic Patch" on Rhodopsin TM5.

Phosphorylation of G protein-coupled receptors (GPCRs) terminates their ability to couple with and activate G proteins by increasing their affinity for arrestins. Unfortunately, detailed information regarding how GPCRs interact with the kinases responsible for their phosphorylation is still limited. Here, we purified fully functional GPCR kinase 1 (GRK1) using a rapid method and used it to gain insights into how this important kinase interacts with the GPCR rhodopsin. Specifically, we find that GRK1 uses the same site on rhodopsin as the transducin (Gt) Gtα C-terminal tail and the arrestin "finger loop", a cleft formed in the cytoplasmic face of the receptor upon activation. Our studies also show GRK1 requires two conserved residues located in this cleft (L226 and V230) that have been shown to be required for Gt activation due to their direct interactions with hydrophobic residues on the Gα C-terminal tail. Our data and modeling studies are consistent with the idea that all three proteins (Gt, GRK1, and visual arrestin) bind, at least in part, in the same site on rhodopsin and interact with the receptor through a similar hydrophobic contact-driven mechanism.

Crystal structure of a common GPCR-binding interface for G protein and arrestin.

G-protein-coupled receptors (GPCRs) transmit extracellular signals to activate intracellular heterotrimeric G proteins (Gαβγ) and arrestins. For G protein signalling, the Gα C-terminus (GαCT) binds to a cytoplasmic crevice of the receptor that opens upon activation. A consensus motif is shared among GαCT from the Gi/Gt family and the ‘finger loop’ region (ArrFL1–4) of all four arrestins. Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin. Functional binding of ArrFL to the receptor was confirmed by ultraviolet-visible absorption spectroscopy, competitive binding assays and Fourier transform infrared spectroscopy. For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice. Our results reflect both the common receptor-binding interface and the divergent biological functions of G proteins and arrestins.

A Transient Interaction between the Phosphate Binding Loop and Switch I Contributes to the Allosteric Network between Receptor and Nucleotide in Gαi1

Receptor-mediated activation of the Gα subunit of heterotrimeric G proteins requires allosteric communication between the receptor binding site and the guanine nucleotide binding site, which are separated by >30 Å. Structural changes in the allosteric network connecting these sites are predicted to be transient in the wild-type Gα subunit, making studies of these connections challenging. In the current work, site-directed mutants that alter the energy barriers between the activation states are used as tools to better understand the transient features of allosteric signaling in the Gα subunit. The observed differences in relative receptor affinity for intact Gαi1 subunits versus C-terminal Gαi1 peptides harboring the K345L mutation are consistent with this mutation modulating the allosteric network in the protein subunit. Measurement of nucleotide exchange rates, affinity for metarhodopsin II, and thermostability suggest that the K345L Gαi1 variant has reduced stability in both the GDP-bound and nucleotide-free states as compared with wild type but similar stability in the GTPγS-bound state. High resolution x-ray crystal structures reveal conformational changes accompanying the destabilization of the GDP-bound state. Of these, the conformation for Switch I was stabilized by an ionic interaction with the phosphate binding loop. Further site-directed mutagenesis suggests that this interaction between Switch I and the phosphate binding loop is important for receptor-mediated nucleotide exchange in the wild-type Gαi1 subunit.

Conformational Dynamics During GPCR - G Protein Coupling

Upon activation by agonists, G-protein coupled receptors (GPCRs) in their active R∗ form transmit extracellular signals into the cell by catalysing GDP/GTP exchange in heterotrimeric G-proteins (Gαsγ, G). The GPCR rhodopsin activates Gt to transmit the light signal into retinal rod cells. After photon capture, the rhodopsin activity jumps by more than one billion fold implying both high fidelity and speed of active rhodopsin (RhR∗)/Gt coupling. We employed all-atom molecular dynamics (MD) simulations to study the conformational diversity of membrane embedded rhodopsin and extend the static picture provided by the available crystal structures. An intrinsically unstructured cytoplasmic loop region connecting transmembrane helices 5 and 6 (ICL3) is identified. The MD data show how each protein state is split into several conformational substates. Only a single conformational substate with largely helical ICL3 is selectively stabilized by the Gtα C-terminus (GtαCT), as in the crystal structures. A mechanism for the fast and precise signal transfer from rhodopsin to Gt is proposed, which assumes a stepwise and mutual reduction of their conformational space. The analysis was extended to the interaction of GsαCT and GiαCT with activated β2-adrenoceptor (β2AR∗) to address the issue of receptor promiscuity. We find that a short region of GsαCT stabilizes the open β2AR∗ conformation provided by the β2AR∗/Gsαsγ crystal structure. In the crystal structures of RhR∗ with GtαCT, a close homologue of GiαCT, RhR∗ exhibits a more closed active conformation. With the slim GiαCT peptide we find that β2AR∗ adopts a similarly closed conformation as RhR∗ with GtαCT. This analysis elucidates how G-protein coupling specificity relies on the fold and primary structure of the Gα C-terminus.Scheerer et al. (2008), Crystal structure of opsin in its G-protein-interacting conformation, NATUREElgeti et al. (2013) Precision vs Flexibility in GPCR signaling, JACS

Linking receptor activation to changes in Sw I and II of Gα proteins

G-protein coupled receptors catalyze nucleotide exchange on G proteins, which results in subunit dissociation and effector activation. In the recent β2AR-Gs structure, portions of Switch I and II of Gα are not fully elucidated. We paired fluorescence studies of receptor-Gαi interactions with the β2AR-Gs and other Gi structures to investigate changes in Switch I and II during receptor activation and GTP binding. The β2/β3 loop containing Leu194 of Gαi is located between Switches I and II, in close proximity to IC2 of the receptor and the C-terminus of Gα, thus providing an allosteric connection between these Switches and receptor activation. We compared the environment of residues in myristoylated Gαi proteins in the heterotrimer to that upon receptor activation and subsequent GTP binding. Upon receptor activation, residues in both Switch regions are less solvent-exposed, as compared to the heterotrimer. Upon GTPγS binding, the environment of several residues in Switch I resemble the receptor-bound state, while Switch II residues display effects on their environment which are consistent with their role in GTP binding and Gβγ dissociation. The ability to merge available crystal structures with solution studies is a powerful tool to gain insight into conformational changes associated with receptor-mediated Gi protein activation.

Dynamics of C-Terminal Gtα and Gsα Peptides in the Binding Cavity of Active GPCRs

On activation by agonists, G protein coupled receptors (GPCRs) transmit extracellular signals into the cell by catalyzing GDP/GTP exchange in heterotrimeric G proteins (Gαsγ). During this reaction the Gα C-terminus (GαCT) that binds to an open binding cavity of active GPCRs is displaced. We report on molecular dynamics simulations of GsαCT and GtαCT peptides interacting with the active GPCRs rhodopsin and β2-adrenoceptor, respectively. Starting from their X-ray structure position, all specific interactions are preserved. When starting from the position in a GDP bound intermediate, obtained from flexible docking, GαCT switches within nanoseconds to the X-ray structure position. Both GαCTs are thereby rotated and expose two distinct sites to highly conserved motifs at R3.50 and at the P3.57 cap of TM3. Our analysis highlights the role of GαCT as an active agent in transferring the signal from the receptor/ G protein interface to the Gα nucleotide binding site.

Hβ2R–Gαs complex: prediction versus crystal structure—how valuable are predictions based on molecular modeling studies?

In 2010, we predicted two models for the hβ(2)R-Gα(s) complex by combining the technique of homology modeling with a potential energy surface scan, since a complete crystal structure of the hβ(2)R-Gα(s) complex was not available. The crystal structure of opsin co-crystallized with part of the C-terminus of Gα (3DQB) was used as a template to model the hβ(2)R, whereas the crystal structure of Gα (1AZT) was used as a template to model Gα(s). Utilizing a potential energy surface scan between hβ(2)R and Gα(s), a six-dimensional potential energy surface was obtained. Two significant minimum regions were located on this surface, and each was associated with a distinct hβ(2)R-Gα(s) complex, namely model I and model II [Straßer A, Wittmann H-J (2010) J Mol Model 16:1307-1318]. The crystal structure of the hβ(2)R-Gα(s)βγ complex has recently been published. Thus, the aim of the current study was, on the one hand, to compare our predicted structures with the true crystal structure, and on the other to discuss the question: how valuable are predictions based on molecular modeling studies?

FTIR Studies On The Activation Mechanism Of Rhodopsin; Possible Extension To General GPCR's

The visual pigment rhodopsin belongs to the group of GPCR's and is so far the best characterized member. Although the structure is known since 2000, the activation mechanism is still not completely understood. Activation is triggered by the light-induced conversion of the covalently bound inverse agonist, 11-cis retinal, to the all-trans geometry, constituting an agonist. We have studied the activation mechanism by Fourier transform infrared difference spectroscopy, a technique capable of monitoring molecular changes of proteins and co-factors. Therefore, this technique could also be of importance for the study of general GPCR's. Some of our recent results will be reviewed. They include the study of the retinal-free opsin state, which can be activated by low pH. This observation has been relevant for the recently published structures of opsin. Next, we show how the technique can be used to study conformational changes of the active state induced by the interacting G-protein (here a peptide derived from the Gα C-terminus known to be responsible for the interaction). In addition, the technique reveals conformational changes of the peptide. Further, the different ionic locks keeping the receptor in its inactive state are addressed. They encompass the Schiff base-counterion, the ERY motif at the end helix C, and the interaction of this motif with two further glutamic acids on helix F. This interaction is thought to keep also the β-adrenergic receptor in its inactive conformation. Finally, a method will be proposed which will allow the study of the ligand-induced activation of general GPCR´s. The essential feature is a receptor monolayer, allowing free access of the ligand. The required increase in sensitivity is realized by surface-enhanced infrared spectroscopy.

Rhodopsin–transducin coupling: Role of the Gα C-terminus in nucleotide exchange catalysis

In the early steps of visual signal transduction, light-activated rhodopsin (R*) catalyzes GDP/GTP exchange in the heterotrimeric G protein (Gαβγ) transducin. We recently reported that the catalytic interaction involves two sequential steps. An initial docking between R* and Gβγ leads to conformational changes which make the C-terminus of Gα (CTα) available for binding to R*. Binding of CTα by R* then triggers GDP/GTP exchange in the Gα subunit. To further study this two-step mechanism, we investigated different single amino acid substitutions within CTα and discuss the effects of high affinity mutations on nucleotide exchange catalysis.

Differential effects of RGS proteins on Gα q and Gα 11 activity

Heterotrimeric G proteins play a pivotal role in GPCR signalling; they link receptors to intracellular effectors and their inactivation by RGS proteins is a key factor in resetting the pathway following stimulation. The precise GPCR:G protein:RGS combination determines the nature and duration of the response. Investigating the activity of particular combinations is difficult in cells which contain multiples of each component. We have therefore utilised a previously characterised yeast system to express mammalian proteins in isolation. Human Gα q and Gα 11 spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of Gα-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both Gα q and Gα 11 but, in contrast, RGS5 and RGS16 were much less effective against Gα 11 than Gα q. Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active Gα q QL/Gα 11 QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-Gα specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of Gα q/Gα 11 enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both Gα subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential Gα activity, further highlighting that RGS proteins can discriminate between two very closely related Gα subunits.

Structural basis of receptor‐dependent G protein activation

Heptahelical receptors activate intracellular signaling pathways by catalyzing GTP/GDP exchange on the α subunit (Gα) of heterotrimeric G proteins. GDP release is rate-limiting for the initiation of all downstream signaling and, until GTP binds, a high-affinity complex between the activated receptor (R*) and nucleotide-free G protein is formed. Despite the important role of the R*-G protein interaction in cell signaling, little is known about the structure of this complex. Biochemical studies suggest the nucleotide binding pocket is nearly 30 Å away from the receptor contacts; how the receptor catalyzes GDP release from this distance is unknown. Over 50 single cysteine substitutions have been introduced into a Gαi1 background to investigate R*-mediated conformational changes using site-directed spin-labeling and electron paramagnetic resonance spectroscopy (EPR). These studies indicate that R* causes a rigid body rotation and translation of the α5 helix, which connects the Gα C-terminus, a well known receptor contact site, to the nucleotide binding pocket at the β 6/α5 loop. No R*-dependent conformational changes have been observed in the N-terminus or Switch II. Double electron-electron resonance EPR distance measurements were made to confirm this movement. This conformational change was not observed when a flexible loop was inserted between the α5 helix and C-terminus to uncouple R*-binding from nucleotide exchange, suggesting that the movement of the α5 helix is necessary for GDP release. Supported by NIH grants EY06062 (H.E.H.), EY005216 (W.L.H.) and GM07347 (W.M.O.), the PhRMA Foundation (W.M.O.) and the Jules Stein Professorship (W.L.H.).

Replacement of the α5 helix of Gα 16 with Gα s-specific sequences enhances promiscuity of Gα 16 toward G s-coupled receptors

G 16 can couple indiscriminately to a large number of G protein-coupled receptors (GPCRs), making it a prime candidate as a universal adaptor for GPCRs. In order to increase the promiscuity of Gα 16, three chimeras incorporating increasing lengths of G s-specific residues (25, 44 or 81 residues) into the C-terminus of Gα 16 were constructed and named 16s25, 16s44 and 16s81, respectively. The chimeras were examined for their ability to mediate receptor-induced stimulation of phospholipase C (PLC) and Ca 2+ mobilization. 16s25 was more effective than 16s44 and 16s81 at coupling to G s-linked receptors. 16s25 coupled productively to 10 different G s-coupled receptors examined and, for 50% of these receptors, 16s25-mediated PLC activities were higher than those mediated via Gα 16. Similar results were observed for agonist-induced Ca 2+ mobilizations. These results show that incorporating the α5 helix of Gα s into Gα 16 can increase the promiscuity of 16s25 towards G s-coupled receptors.

Regulation of adenylyl cyclase from Blastocladiella emersonii by guanine nucleotides

GTPγS stimulates adenylyl cyclase in particulate fractions of Blastocladiella emersonii zoospores. Cholera toxin catalyses the ADP-ribosylation of a membrane protein of a molecular weight (46,000) similar to that of the α subunit of G s found in vertebrate cells. A membrane protein of 46 kDa can also be recognized in Western blots by an antipeptide antiserum (RM/1) raised against the C-terminus of Gα s -subunits. These results suggest that a G-protein mediates the regulation of Blastocladiella adenylyl cyclase by guanine nucleotides.