Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.

Heterotrimeric G protein signaling without GPCRs: The Gα-binding-and-activating (GBA) motif.

Heterotrimeric G proteins (Gαßγ) are molecular switches that relay signals from 7-transmembrane receptors located at the cell surface to the cytoplasm. The function of these receptors is so intimately linked to heterotrimeric G proteins that they are named G protein-coupled receptors (GPCRs), showcasing the interdependent nature of this archetypical receptor-transducer axis of transmembrane signaling in eukaryotes. It is generally assumed that activation of heterotrimeric G protein signaling occurs exclusively by the action of GPCRs, but this idea has been challenged by the discovery of alternative mechanisms by which G proteins can propagate signals in the cell. This review will focus on a general principle of G protein signaling that operates without the direct involvement of GPCRs. The mechanism of G protein signaling reviewed here is mediated by a class of G protein regulators defined by containing an evolutionarily conserved sequence named the Gα-binding-and-activating (GBA) motif. Using the best characterized proteins with a GBA motif as examples, Gα-interacting vesicle-associated protein (GIV)/Girdin and dishevelled-associating protein with a high frequency of leucine residues (DAPLE), this review will cover (i) the mechanisms by which extracellular cues not relayed by GPCRs promote the coupling of GBA motif-containing regulators with G proteins, (ii) the structural and molecular basis for how GBA motifs interact with Gα subunits to facilitate signaling, (iii) the relevance of this mechanism in different cellular and pathological processes, including cancer and birth defects, and (iv) strategies to manipulate GBA-G protein coupling for experimental therapeutics purposes, including the development of rationally engineered proteins and chemical probes.

Dissecting the molecular basis for the modulation of neurotransmitter GPCR signaling by GINIP.

It is well established that G-protein-coupled receptors (GPCRs) stimulated by neurotransmitters are critical for neuromodulation. Much less is known about how heterotrimeric G-protein (Gαßγ) regulation after receptor-mediated activation contributes to neuromodulation. Recent evidence indicates that the neuronal protein GINIP shapes GPCR inhibitory neuromodulation via a unique mechanism of G-protein regulation that controls pain and seizure susceptibility. However, the molecular basis of this mechanism remains ill-defined because the structural determinants of GINIP responsible for binding and regulating G proteins are not known. Here, we combined hydrogen-deuterium exchange mass spectrometry, computational structure predictions, biochemistry, and cell-based biophysical assays to demonstrate an effector-like binding mode of GINIP to Gαi. Specific amino acids of GINIP's PHD domain first loop are essential for G-protein binding and subsequent regulation of Gαi-GTP and Gßγ signaling upon neurotransmitter GPCR stimulation. In summary, these findings shed light onto the molecular basis for a post-receptor mechanism of G-protein regulation that fine-tunes inhibitory neuromodulation.

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.

CaaX-motif-adjacent residues influence G protein gamma (Gγ) prenylation under suboptimal conditions.

Prenylation is an irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Dysregulation of prenylation contributes to multiple disorders, including cancers and vascular and neurodegenerative diseases. Prenyltransferases tether isoprenoid lipids to proteins via a thioether linkage during prenylation. Pharmacological inhibition of the lipid synthesis pathway by statins is a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential inhibition. We examined the prenylation of carboxy-terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to prenylation and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide, statin sensitivity, and extent of prenylation. Our results also show a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings indicate a plausible mechanism allowing for statins to differentially perturb heterotrimeric G protein signaling in cells depending on their Gγ-subtype composition. Our results may also provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.

Ligand and G-protein selectivity in the κ-opioid receptor.

The κ-opioid receptor (KOR) represents a highly desirable therapeutic target for treating not only pain but also addiction and affective disorders1. However, the development of KOR analgesics has been hindered by the associated hallucinogenic side effects2. The initiation of KOR signalling requires the Gi/o-family proteins including the conventional (Gi1, Gi2, Gi3, GoA and GoB) and nonconventional (Gz and Gg) subtypes. How hallucinogens exert their actions through KOR and how KOR determines G-protein subtype selectivity are not well understood. Here we determined the active-state structures of KOR in a complex with multiple G-protein heterotrimers-Gi1, GoA, Gz and Gg-using cryo-electron microscopy. The KOR-G-protein complexes are bound to hallucinogenic salvinorins or highly selective KOR agonists. Comparisons of these structures reveal molecular determinants critical for KOR-G-protein interactions as well as key elements governing Gi/o-family subtype selectivity and KOR ligand selectivity. Furthermore, the four G-protein subtypes display an intrinsically different binding affinity and allosteric activity on agonist binding at KOR. These results provide insights into the actions of opioids and G-protein-coupling specificity at KOR and establish a foundation to examine the therapeutic potential of pathway-selective agonists of KOR.

Ultrafast structural changes direct the first molecular events of vision.

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

The role of G protein conformation in receptor-G protein selectivity.

G protein-coupled receptors (GPCRs) selectively activate at least one of the four families of heterotrimeric G proteins, but the mechanism of coupling selectivity remains unclear. Structural studies emphasize structural complementarity of GPCRs and nucleotide-free G proteins, but selectivity is likely to be determined by transient intermediate-state complexes that exist before nucleotide release. Here we study coupling to nucleotide-decoupled G protein variants that can adopt conformations similar to receptor-bound G proteins without releasing nucleotide, and are therefore able to bypass intermediate-state complexes. We find that selectivity is degraded when nucleotide release is not required for GPCR-G protein complex formation, to the extent that most GPCRs interact with most nucleotide-decoupled G proteins. These findings demonstrate the absence of absolute structural incompatibility between noncognate receptor-G protein pairs, and are consistent with the hypothesis that transient intermediate states are partly responsible for coupling selectivity.

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.

R4 RGS proteins as fine tuners of immature and mature hematopoietic cell trafficking.

G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors. They are involved in almost every physiologic process and consequently have a pivotal role in an extensive number of pathologies, including genetic, neurologic, and immune system disorders. Indeed, the vast array of GPCRs mechanisms have led to the development of a tremendous number of drug therapies and already account for about a third of marketed drugs. These receptors mediate their downstream signals primarily via G proteins. The regulators of G-protein signaling (RGS) proteins are now in the spotlight as the critical modulatory factors of active GTP-bound Gα subunits of heterotrimeric G proteins to fine-tune the biologic responses driven by the GPCRs. Also, they possess noncanonical functions by multiple mechanisms, such as protein-protein interactions. Essential roles and impacts of these RGS proteins have been revealed in physiology, including hematopoiesis and immunity, and pathologies, including asthma, cancers, and neurologic disorders. This review focuses on the largest subfamily of R4 RGS proteins and provides a brief overview of their structures and G-proteins selectivity. With particular interest, we explore and highlight, their expression in the hematopoietic system and the regulation in the engraftment of hematopoietic stem/progenitor cells (HSPCs). Distinct expression patterns of R4 RGS proteins in the hematopoietic system and their pivotal roles in stem cell trafficking pave the way for realizing new strategies for enhancing the clinical performance of hematopoietic stem cell transplantation. Finally, we discuss the exciting future trends in drug development by targeting RGS activity and expression with small molecules inhibitors and miRNA approaches.

PRECOGx: exploring GPCR signaling mechanisms with deep protein representations.

In this study we show that protein language models can encode structural and functional information of GPCR sequences that can be used to predict their signaling and functional repertoire. We used the ESM1b protein embeddings as features and the binding information known from publicly available studies to develop PRECOGx, a machine learning predictor to explore GPCR interactions with G protein and ß-arrestin, which we made available through a new webserver (https://precogx.bioinfolab.sns.it/). PRECOGx outperformed its predecessor (e.g. PRECOG) in predicting GPCR-transducer couplings, being also able to consider all GPCR classes. The webserver also provides new functionalities, such as the projection of input sequences on a low-dimensional space describing essential features of the human GPCRome, which is used as a reference to track GPCR variants. Additionally, it allows inspection of the sequence and structural determinants responsible for coupling via the analysis of the most important attention maps used by the models as well as through predicted intramolecular contacts. We demonstrate applications of PRECOGx by predicting the impact of disease variants (ClinVar) and alternative splice forms from healthy tissues (GTEX) of human GPCRs, revealing the power to dissect system biasing mechanisms in both health and disease.

Heterotrimeric G Protein α-Subunits - Structures, Peptide-Derived Inhibitors, and Mechanisms.

G protein-coupled receptors are the largest protein family in the human body and represent the most important class of drug targets. They receive extracellular signals and transduce them into the cytosol. The guanine nucleotide-binding Gα proteins represent the main relays by which GPCRs induce intracellular effects. More than 800 different GPCRs interact with 16 Gα proteins belonging to 4 families, Gαi, Gαs, Gαq, and Gα12/13. The direct inhibition of Gα protein subunits rather than the modulation of GPCR subtypes has been proposed as a novel strategy for the treatment of complex diseases, including inflammation and cancer. This mini-review presents an introduction to G protein structure and function and describes achievements in the development of peptidic and peptide-derived Gα protein inhibitors. They have become indispensable pharmacological tools, and some of them exhibit significant potential as future drugs.

Effective Strategies for Heterologous Expression of Plant Heterotrimeric G-protein γ Subunits without Gß Subunit Partners.

BACKGROUND: In plants, heterotrimeric G-protein (Gγ) subunits are diverse, and they have structural plasticity to provide functional selectivity to the heterotrimer. Although the Gß and Gγ subunits dimerize to function in the signaling pathway, the interaction mechanism of various Gγ subunits with the Gß subunit partners is still elusive. OBJECTIVE: To better understand the interaction mechanism, one approach is to separate the subunits for the re-assembly in vitro. Hence, developing a reliable method for achieving the efficient production and purification of these proteins has become necessary. METHODS: In this study, Gγ1 and Gγ2 proteins from Oryza sativa and Arabidopsis thaliana were successfully identified, cloned, expressed in bacteria, and purified as recombinant proteins with the fusion tags. Highly expressed recombinant Gγ subunits in E. coli were digested by proteases, which were also produced in the presented study. RESULTS: Preliminary structural characterization studies without the Gß partners showed that Gγ1 proteins have disordered structures with coiled-coil, α-helix extensions, and loops, whereas the Gγ2 protein has a more dominant ß-sheet and turns structure. Finally, computational analyses performed on Gγ genes have laid the foundation of new targets for biotechnological purposes. CONCLUSION: The proposed optimized expression and purification protocol can contribute to investigations on the Gßγ binding mechanism in plant G-protein signaling. The investigations on selective binding are critical to shed light on the role(s) of different plant Gγ subunit types in biological processes.

G-protein activation by a metabotropic glutamate receptor.

Family C G-protein-coupled receptors (GPCRs) operate as obligate dimers with extracellular domains that recognize small ligands, leading to G-protein activation on the transmembrane (TM) domains of these receptors by an unknown mechanism1. Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric Gi. Upon activation of the extracellular domain, the two transmembrane domains undergo extensive rearrangement in relative orientation to establish an asymmetric TM6-TM6 interface that promotes conformational changes in the cytoplasmic domain of one protomer. Nucleotide-bound Gi can be observed pre-coupled to inactive mGlu2, but its transition to the nucleotide-free form seems to depend on establishing the active-state TM6-TM6 interface. In contrast to family A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6 but is facilitated through the coordination of intracellular loops 2 and 3, as well as a critical contribution from the C terminus of the receptor. The findings highlight the synergy of global and local conformational transitions to facilitate a new mode of G-protein activation.

GPCRsignal: webserver for analysis of the interface between G-protein-coupled receptors and their effector proteins by dynamics and mutations.

GPCRsignal (https://gpcrsignal.biomodellab.eu/) is a webserver devoted to signaling complexes of G-protein-coupled receptors (GPCRs). The recent improvement in cryo-electron microscopy resulted in the determination of a large number of high-resolution structures of GPCRs bound to their effector proteins: G proteins or arrestins. Analyzing the interfaces between receptor and an effector protein is of high importance since a selection of proper G protein or specific conformation of arrestin leads to changes of signaling that can significantly affect action of drugs. GPCRsignal provides a possibility of running molecular dynamics simulations of all currently available GPCR-effector protein complexes for curated structures: wild-type, with crystal/cryo-EM mutations, or with mutations introduced by the user. The simulations are performed in an implicit water-membrane environment, so they are rather fast. User can run several simulations to obtain statistically valid results. The simulations can be analyzed separately using dynamic FlarePlots for particular types of interactions. One can also compare groups of simulations in Interaction frequency analysis as HeatMaps and also in interaction frequency difference analysis as sticks, linking the interacting residues, of different color and size proportional to differences in contact frequencies.

Structural insights into the lipid and ligand regulation of serotonin receptors.

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest subtype family of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1E and other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor-G-protein complexes: 5-HT1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1D bound to 5-HT; and 5-HT1E in complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein-5-HT1A interface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules-particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.

Delineating the conformational landscape of the adenosine A2A receptor during G protein coupling.

G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are important drug targets. Their diverse signaling pathways are driven by complex pharmacology arising from a conformational ensemble rarely captured by structural methods. Here, fluorine nuclear magnetic resonance spectroscopy (19F NMR) is used to delineate key functional states of the adenosine A2A receptor (A2AR) complexed with heterotrimeric G protein (Gαsß1γ2) in a phospholipid membrane milieu. Analysis of A2AR spectra as a function of ligand, G protein, and nucleotide identifies an ensemble represented by inactive states, a G-protein-bound activation intermediate, and distinct nucleotide-free states associated with either partial- or full-agonist-driven activation. The Gßγ subunit is found to be critical in facilitating ligand-dependent allosteric transmission, as shown by 19F NMR, biochemical, and computational studies. The results provide a mechanistic basis for understanding basal signaling, efficacy, precoupling, and allostery in GPCRs.

Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs.

G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1ß1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.

A universal allosteric mechanism for G protein activation.

G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote guanosine triphosphate (GTP) binding and dissociation of Gα from the Gßγ subunits. Structural studies have revealed the molecular basis of subunit association with receptors, RGS proteins, and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We use cell signaling assays, molecular dynamics (MD) simulations, and biochemistry and structural analyses to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the Gßγ dimer. Replacement of network residues prevents subunit dissociation regardless of agonist or GTP binding. These findings reveal the molecular basis of the final committed step of G protein activation.

Structure of the class D GPCR Ste2 dimer coupled to two G proteins.

G-protein-coupled receptors (GPCRs) are divided phylogenetically into six classes1,2, denoted A to F. More than 370 structures of vertebrate GPCRs (belonging to classes A, B, C and F) have been determined, leading to a substantial understanding of their function3. By contrast, there are no structures of class D GPCRs, which are found exclusively in fungi where they regulate survival and reproduction. Here we determine the structure of a class D GPCR, the Saccharomyces cerevisiae pheromone receptor Ste2, in an active state coupled to the heterotrimeric G protein Gpa1-Ste4-Ste18. Ste2 was purified as a homodimer coupled to two G proteins. The dimer interface of Ste2 is formed by the N terminus, the transmembrane helices H1, H2 and H7, and the first extracellular loop ECL1. We establish a class D1 generic residue numbering system (CD1) to enable comparisons with orthologues and with other GPCR classes. The structure of Ste2 bears similarities in overall topology to class A GPCRs, but the transmembrane helix H4 is shifted by more than 20 Å and the G-protein-binding site is a shallow groove rather than a cleft. The structure provides a template for the design of novel drugs to target fungal GPCRs, which could be used to treat numerous intractable fungal diseases4.