Instructor, Molecular & Cellular Physiology
Structures of Ric-8B in complex with Galpha protein folding clients reveal isoform specificity mechanisms.
Structure (London, England : 1993)
Mammalian Ric-8 proteins act as chaperones to regulate the cellular abundance of heterotrimeric G protein alpha subunits. The Ric-8A isoform chaperones Galphai/o, Galpha12/13, and Galphaq/11 subunits, while Ric-8B acts on Galphas/olf subunits. Here, we determined cryoelectron microscopy (cryo-EM) structures of Ric-8B in complex with Galphas and Galphaolf, revealing isoform differences in the relative positioning and contacts between the C-terminal alpha5 helix of Galpha within the concave pocket formed by Ric-8 alpha-helical repeat elements. Despite the overall architectural similarity with our earlier structures of Ric-8A complexed to Galphaq and Galphai1, Ric-8B distinctly accommodates an extended loop found only in Galphas/olf proteins. The structures, along with results from Ric-8 protein thermal stability assays and cell-based Galphaolf folding assays, support a requirement for the Galpha C-terminal region for binding specificity, and highlight that multiple structural elements impart specificity for Ric-8/G protein binding.
View details for DOI 10.1016/j.str.2023.02.011
View details for PubMedID 36931277
Structure determination of inactive-state GPCRs with a universal nanobody.
Nature structural & molecular biology
Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. Despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that the same single-chain camelid antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained structures of neurotensin 1 receptor bound to antagonist SR48692, mu-opioid receptor bound to alvimopan, apo somatostatin receptor 2 and histamine receptor 2 bound to famotidine. We expect this rapid, straightforward approach to facilitate the broad exploration of GPCR inactive states without the need for extensive engineering and crystallization.
View details for DOI 10.1038/s41594-022-00859-8
View details for PubMedID 36396979
The tethered peptide activation mechanism of adhesion GPCRs.
Adhesion G-protein-coupled receptors (aGPCRs) are characterized by the presence of auto-proteolysing extracellular regions that are involved in cell-cell and cell-extracellular matrix interactions1. Self cleavage within the aGPCR auto-proteolysis-inducing (GAIN) domain produces two protomers-N-terminal and C-terminal fragments-that remain non-covalently attached after receptors reach the cell surface1. Upon dissociation of the N-terminal fragment, the C-terminus of the GAIN domain acts as a tethered agonist (TA) peptide to activate the seven-transmembrane domain with a mechanism that has been poorly understood2-5. Here we provide cryo-electron microscopy snapshots of two distinct members of the aGPCR family, GPR56 (also known as ADGRG1) and latrophilin3 (LPHN3 (also known as ADGRL3)). Low-resolution maps of the receptors in their N-terminal fragment-bound state indicate that the GAIN domain projects flexibly towards the extracellular space, keeping the encrypted TA peptide away from the seven-transmembrane domain. High-resolution structures of GPR56 and LPHN3 in their active, G-protein-coupled states, reveal that after dissociation of the extracellular region, the decrypted TA peptides engage the seven-transmembrane domain core with a notable conservation of interactions that also involve extracellular loop 2. TA binding stabilizes breaks in the middle of transmembrane helices 6 and 7 that facilitate aGPCR coupling and activation of heterotrimeric G proteins. Collectively, these results enable us to propose a general model for aGPCR activation.
View details for DOI 10.1038/s41586-022-04575-7
View details for PubMedID 35418682
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.
View details for DOI 10.1038/s41586-021-03680-3
View details for PubMedID 34194039
Structures of Gα Proteins in Complex with Their Chaperone Reveal Quality Control Mechanisms.
Many chaperones promote nascent polypeptide folding followed by substrate release through ATP-dependent conformational changes. Here we show cryoEM structures of Gα subunit folding intermediates in complex with full-length Ric-8A, a unique chaperone-client system in which substrate release is facilitated by guanine nucleotide binding to the client G protein. The structures of Ric-8A-Gαi and Ric-8A-Gαq complexes reveal that the chaperone employs its extended C-terminal region to cradle the Ras-like domain of Gα, positioning the Ras core in contact with the Ric-8A core while engaging its switch2 nucleotide binding region. The C-terminal α5 helix of Gα is held away from the Ras-like domain through Ric-8A core domain interactions, which critically depend on recognition of the Gα C terminus by the chaperone. The structures, complemented with biochemical and cellular chaperoning data, support a folding quality control mechanism that ensures proper formation of the C-terminal α5 helix before allowing GTP-gated release of Gα from Ric-8A.
View details for DOI 10.1016/j.celrep.2020.02.086
View details for PubMedID 32126208
Structures of metabotropic GABAB receptor.
GABA (γ-aminobutyric acid) stimulation of the metabotropic GABAB receptor results in prolonged inhibition of neurotransmission that is central to brain physiology1. GABAB belongs to the Family C of G protein-coupled receptors (GPCRs), which operate as dimers to relay synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins2,3. GABAB, however, is unique in its function as an obligate heterodimer in which agonist binding and G protein activation take place on distinct subunits4,5. Here we show structures of heterodimeric and homodimeric full-length GABAB receptors. Complemented by cellular signaling assays and atomistic simulations, the structures reveal an essential role for the GABAB extracellular loop 2 (ECL2) in relaying structural transitions by ordering the linker connecting the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of both GABAB subunits caps and interacts with the hydrophilic head of a phospholipid occupying the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G protein. These results provide a starting framework to decipher mechanistic modes of signal transduction mediated by GABAB dimers and have important implications for rational drug design targeting these receptors.
View details for DOI 10.1038/s41586-020-2469-4
View details for PubMedID 32580208
Production of Phosphorylated Ric-8A proteins using protein kinase CK2
PROTEIN EXPRESSION AND PURIFICATION
2019; 154: 98–103
Resistance to Inhibitors of Cholinesterase-8 (Ric-8) proteins are molecular chaperones that fold heterotrimeric G protein α subunits shortly after biosynthesis. Ric-8 proteins also act as test tube guanine nucleotide exchange factors (GEF) that promote Gα subunit GDP for GTP exchange. The GEF and chaperoning activities of Ric-8A are regulated by phosphorylation of five serine and threonine residues within protein kinase CK2 consensus sites. The traditional way that Ric-8A proteins have been purified is from Spodoptera frugiperda (Sf9) or Trichoplusia ni (Tni) insect cells. Endogenous insect cell kinases do phosphorylate the critical regulatory sites of recombinant Ric-8A reasonably well, but there is batch-to-batch variability among recombinant Ric-8A preparations. Additionally, insect cell-production of some Ric-8 proteins with phosphosite alanine substitution mutations is proscribed as there seems to be interdependency of multi-site phosphorylation for functional protein production. Here, we present a method to produce wild type and phosphosite mutant Ric-8A proteins that are fully occupied with bound phosphate at each of the regulatory positions. Ric-8A proteins were expressed and purified from E. coli. Purified Ric-8A was phosphorylated in vitro with protein kinase CK2 and then re-isolated to remove kinase. The phosphorylated Ric-8A proteins were ∼99% pure and the completeness of phosphorylation was verified by chromatography, phos-tag SDS-PAGE mobility shifts, immunoblotting using phospho-site specific antibodies, and mass spectrometry analysis. E. coli-produced Ric-8A that was phosphorylated using this method promoted a faster rate of Gα subunit guanine nucleotide exchange than Ric-8A that was variably phosphorylated during production in insect cells.
View details for DOI 10.1016/j.pep.2018.10.002
View details for Web of Science ID 000451654000013
View details for PubMedID 30290220
View details for PubMedCentralID PMC6240494
Structure, Function, and Dynamics of the Gα Binding Domain of Ric-8A.
Structure (London, England : 1993)
Ric-8A is a 530-amino acid cytoplasmic molecular chaperone and guanine nucleotide exchange factor (GEF) for i, q, and 12/13 classes of heterortrimeric G protein alpha subunits (Gα). We report the 2.2-Å crystal structure of the Ric-8A Gα-binding domain with GEF activity, residues 1-452, and is phosphorylated at Ser435 and Thr440. Residues 1-429 adopt a superhelical fold comprised of Armadillo (ARM) and HEAT repeats, and the C terminus is disordered. One of the phosphorylated residues potentially binds to a basic cluster in an ARM motif. Amino acid sequence conservation and published hydrogen-deuterium exchange data indicate repeats 3 through 6 to be a putative Gα-binding surface. Normal mode modeling of small-angle X-ray scattering data indicates that phosphorylation induces relative rotation between repeats 1-4, 5-6, and 7-9. 2D 1H-15N-TROSY spectra of [2H,15N]-labeled Gαi1 in the presence of R452 reveals chemical shift perturbations of the C terminus and Gαi1 residues involved in nucleotide binding.
View details for DOI 10.1016/j.str.2019.04.013
View details for PubMedID 31155309
Dual phosphorylation of Ric-8A enhances its ability to mediate G protein alpha subunit folding and to stimulate guanine nucleotide exchange
2018; 11 (532)
Resistance to inhibitors of cholinesterase-8A (Ric-8A) and Ric-8B are essential biosynthetic chaperones for heterotrimeric G protein α subunits. We provide evidence for the direct regulation of Ric-8A cellular activity by dual phosphorylation. Using proteomics, Western blotting, and mutational analyses, we determined that Ric-8A was constitutively phosphorylated at five serines and threonines by the protein kinase CK2. Phosphorylation of Ser435 and Thr440 in rat Ric-8A (corresponding to Ser436 and Thr441 in human Ric-8A) was required for high-affinity binding to Gα subunits, efficient stimulation of Gα subunit guanine nucleotide exchange, and mediation of Gα subunit folding. The CK2 consensus sites that contain Ser435 and Thr440 are conserved in Ric-8 homologs from worms to mammals. We found that the homologous residues in mouse Ric-8B, Ser468 and Ser473, were also phosphorylated. Mutation of the genomic copy of ric-8 in Caenorhabditis elegans to encode alanine in the homologous sites resulted in characteristic ric-8 reduction-of-function phenotypes that are associated with defective Gq and Gs signaling, including reduced locomotion and defective egg laying. The C. elegans ric-8 phosphorylation site mutant phenotypes were partially rescued by chemical stimulation of Gq signaling. These results indicate that dual phosphorylation represents a critical form of conserved Ric-8 regulation and demonstrate that Ric-8 proteins are needed for effective Gα signaling. The position of the CK2-phosphorylated sites within a structural model of Ric-8A reveals that these sites contribute to a key acidic and negatively charged surface that may be important for its interactions with Gα subunits.
View details for DOI 10.1126/scisignal.aap8113
View details for Web of Science ID 000433426900003
View details for PubMedID 29844055
The G Protein alpha Chaperone Ric-8 as a Potential Therapeutic Target
2015; 87 (1): 52–63
Resistance to inhibitors of cholinesterase (Ric-8)A and Ric-8B are essential genes that encode positive regulators of heterotrimeric G protein α subunits. Controversy persists surrounding the precise way(s) that Ric-8 proteins affect G protein biology and signaling. Ric-8 proteins chaperone nucleotide-free Gα-subunit states during biosynthetic protein folding prior to G protein heterotrimer assembly. In organisms spanning the evolutionary window of Ric-8 expression, experimental perturbation of Ric-8 genes results in reduced functional abundances of G proteins because G protein α subunits are misfolded and degraded rapidly. Ric-8 proteins also act as Gα-subunit guanine nucleotide exchange factors (GEFs) in vitro. However, Ric-8 GEF activity could strictly be an in vitro phenomenon stemming from the ability of Ric-8 to induce partial Gα unfolding, thereby enhancing GDP release. Ric-8 GEF activity clearly differs from the GEF activity of G protein-coupled receptors (GPCRs). G protein βγ is inhibitory to Ric-8 action but obligate for receptors. It remains an open question whether Ric-8 has dual functions in cells and regulates G proteins as both a molecular chaperone and GEF. Clearly, Ric-8 has a profound influence on heterotrimeric G protein function. For this reason, we propose that Ric-8 proteins are as yet untested therapeutic targets in which pharmacological inhibition of the Ric-8/Gα protein-protein interface could serve to attenuate the effects of disease-causing G proteins (constitutively active mutants) and/or GPCR signaling. This minireview will chronicle the understanding of Ric-8 function, provide a comparative discussion of the Ric-8 molecular chaperoning and GEF activities, and support the case for why Ric-8 proteins should be considered potential targets for development of new therapies.
View details for DOI 10.1124/mol.114.094664
View details for Web of Science ID 000346187500006
View details for PubMedID 25319541
View details for PubMedCentralID PMC4279082