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• W3010123279 abstract "•CryoEM structures of Gαi1 and Gαq protein subunits in complex with chaperone Ric-8A•Ric-8 forms a cradle to accommodate the Ras-like domain of Gα•Gα C terminus binding to Ric-8 is a prerequisite for GTP-gated Gα release•Insights into G protein subtype binding specificity by Ric-8 isoforms 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. 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. Heterotrimeric G proteins, composed of Gα, Gβ, and Gγ subunits, relay the vast majority of intracellular signaling mediated by G protein-coupled receptors (GPCRs), the largest and most diverse class of membrane proteins in eukaryotes. GPCRs respond to a remarkable array of extracellular stimulants, including light, ions, hormones, odorants, neurotransmitters, and natural chemicals, and in turn couple primarily to G proteins to facilitate the exchange of GDP for GTP on the Gα subunit (Hilger et al., 2018Hilger D. Masureel M. Kobilka B.K. Structure and dynamics of GPCR signaling complexes.Nat. Struct. Mol. Biol. 2018; 25: 4-12Crossref PubMed Scopus (282) Google Scholar). Nucleotide exchange promotes the functional dissociation of Gα-GTP from the Gβγ obligate dimer, leading to downstream signaling through binding to effectors such as adenylyl cyclase, phospholipase C, and ion channels. G proteins are classified based on sequence homology of the Gα subunit into four main sub-classes: Gαi/o, Gαq/11, Gαs/olf, and Gα12/13, each comprised of multiple isoforms (Wilkie et al., 1992Wilkie T.M. Gilbert D.J. Olsen A.S. Chen X.N. Amatruda T.T. Korenberg J.R. Trask B.J. de Jong P. Reed R.R. Simon M.I. et al.Evolution of the mammalian G protein alpha subunit multigene family.Nat. Genet. 1992; 1: 85-91Crossref PubMed Scopus (206) Google Scholar). Accordingly, GPCRs display distinct selectivity profiles for heterotrimeric G proteins, thereby orchestrating precise cellular pathways and signaling outcomes. The central signaling role of heterotrimeric G proteins, along with their ability to undergo extensive conformational changes during highly tuned receptor coupling and dissociation, has co-evolved with quality control mechanisms for their structural and functional integrity. Recent work has shown that multiple chaperones are necessary for proper folding, assembly, and localization of heterotrimeric G proteins. Chaperonin-containing tailless complex polypeptide-1 (CCT) (Lukov et al., 2005Lukov G.L. Hu T. McLaughlin J.N. Hamm H.E. Willardson B.M. Phosducin-like protein acts as a molecular chaperone for G protein betagamma dimer assembly.EMBO J. 2005; 24: 1965-1975Crossref PubMed Scopus (72) Google Scholar, Lukov et al., 2006Lukov G.L. Baker C.M. Ludtke P.J. Hu T. Carter M.D. Hackett R.A. Thulin C.D. Willardson B.M. Mechanism of assembly of G protein betagamma subunits by protein kinase CK2-phosphorylated phosducin-like protein and the cytosolic chaperonin complex.J. Biol. Chem. 2006; 281: 22261-22274Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and dopamine receptor-interacting protein 78 (DRiP78) (Dupré et al., 2007Dupré D.J. Robitaille M. Richer M. Ethier N. Mamarbachi A.M. Hébert T.E. Dopamine receptor-interacting protein 78 acts as a molecular chaperone for Ggamma subunits before assembly with Gbeta.J. Biol. Chem. 2007; 282: 13703-13715Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) were proposed to facilitate the folding of Gβ and Gγ subunits, respectively, while the chaperone phosducin-like protein-1 (PhLP-1) is subsequently involved in the formation of the Gβγ obligate heterodimer (Lukov et al., 2005Lukov G.L. Hu T. McLaughlin J.N. Hamm H.E. Willardson B.M. Phosducin-like protein acts as a molecular chaperone for G protein betagamma dimer assembly.EMBO J. 2005; 24: 1965-1975Crossref PubMed Scopus (72) Google Scholar, Lukov et al., 2006Lukov G.L. Baker C.M. Ludtke P.J. Hu T. Carter M.D. Hackett R.A. Thulin C.D. Willardson B.M. Mechanism of assembly of G protein betagamma subunits by protein kinase CK2-phosphorylated phosducin-like protein and the cytosolic chaperonin complex.J. Biol. Chem. 2006; 281: 22261-22274Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Resistance to inhibitors of cholinesterase-8 (Ric-8) was initially discovered as a gene that positively influenced G protein signaling pathways in a Caenorhabditis elegans mutagenesis screen (Miller et al., 1996Miller K.G. Alfonso A. Nguyen M. Crowell J.A. Johnson C.D. Rand J.B. A genetic selection for Caenorhabditis elegans synaptic transmission mutants.Proc. Natl. Acad. Sci. USA. 1996; 93: 12593-12598Crossref PubMed Scopus (322) Google Scholar, Nguyen et al., 1995Nguyen M. Alfonso A. Johnson C.D. Rand J.B. Caenorhabditis elegans mutants resistant to inhibitors of acetylcholinesterase.Genetics. 1995; 140: 527-535Crossref PubMed Google Scholar). Ric-8 proteins are now known to fold nascent Gα subunits prior to G protein heterotrimer formation. The initial in vitro work identified Ric-8 as a non-receptor guanine exchange factor (GEF) for Gα proteins (Chan et al., 2011bChan P. Gabay M. Wright F.A. Tall G.G. Ric-8B is a GTP-dependent G protein alphas guanine nucleotide exchange factor.J. Biol. Chem. 2011; 286: 19932-19942Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, Tall et al., 2003Tall G.G. Krumins A.M. Gilman A.G. Mammalian Ric-8A (synembryn) is a heterotrimeric Galpha protein guanine nucleotide exchange factor.J. Biol. Chem. 2003; 278: 8356-8362Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), but more recent studies revealed that Ric-8 proteins are molecular chaperones for nascent Gα subunits (Chan et al., 2013Chan P. Thomas C.J. Sprang S.R. Tall G.G. Molecular chaperoning function of Ric-8 is to fold nascent heterotrimeric G protein α subunits.Proc. Natl. Acad. Sci. USA. 2013; 110: 3794-3799Crossref PubMed Scopus (50) Google Scholar, Gabay et al., 2011Gabay M. Pinter M.E. Wright F.A. Chan P. Murphy A.J. Valenzuela D.M. Yancopoulos G.D. Tall G.G. Ric-8 proteins are molecular chaperones that direct nascent G protein α subunit membrane association.Sci. Signal. 2011; 4: ra79Crossref PubMed Scopus (73) Google Scholar), thereby explaining the positive influence of their activities on G protein signaling (Papasergi et al., 2015Papasergi M.M. Patel B.R. Tall G.G. The G protein α chaperone Ric-8 as a potential therapeutic target.Mol. Pharmacol. 2015; 87: 52-63Crossref PubMed Scopus (24) Google Scholar). Besides facilitating Gα folding, the observed Ric-8 GEF activity for Gα subunits (Chan et al., 2011aChan P. Gabay M. Wright F.A. Kan W. Oner S.S. Lanier S.M. Smrcka A.V. Blumer J.B. Tall G.G. Purification of heterotrimeric G protein alpha subunits by GST-Ric-8 association: primary characterization of purified G alpha(olf).J. Biol. Chem. 2011; 286: 2625-2635Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, Tall et al., 2003Tall G.G. Krumins A.M. Gilman A.G. Mammalian Ric-8A (synembryn) is a heterotrimeric Galpha protein guanine nucleotide exchange factor.J. Biol. Chem. 2003; 278: 8356-8362Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, Van Eps et al., 2015Van Eps N. Thomas C.J. Hubbell W.L. Sprang S.R. The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1.Proc. Natl. Acad. Sci. USA. 2015; 112: 1404-1409Crossref PubMed Scopus (17) Google Scholar) has raised the possibility that these chaperones may also be involved in alternative modes of Gα subunit activation or reamplification of GPCR signaling. Ric-8 proteins are evolutionarily conserved from fungi to humans (Li et al., 2010Li Y. Yan X. Wang H. Liang S. Ma W.B. Fang M.Y. Talbot N.J. Wang Z.Y. MoRic8 Is a novel component of G-protein signaling during plant infection by the rice blast fungus Magnaporthe oryzae.Mol. Plant Microbe Interact. 2010; 23: 317-331Crossref PubMed Scopus (31) Google Scholar, Miller et al., 2000Miller K.G. Emerson M.D. McManus J.R. Rand J.B. RIC-8 (Synembryn): a novel conserved protein that is required for G(q)alpha signaling in the C. elegans nervous system.Neuron. 2000; 27: 289-299Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, Papasergi et al., 2015Papasergi M.M. Patel B.R. Tall G.G. The G protein α chaperone Ric-8 as a potential therapeutic target.Mol. Pharmacol. 2015; 87: 52-63Crossref PubMed Scopus (24) Google Scholar, Wright et al., 2011Wright S.J. Inchausti R. Eaton C.J. Krystofova S. Borkovich K.A. RIC8 is a guanine-nucleotide exchange factor for Galpha subunits that regulates growth and development in Neurospora crassa.Genetics. 2011; 189: 165-176Crossref PubMed Scopus (25) Google Scholar), although no homologs have been reported in plants and baker’s yeast. However, Arr4/Get3 proteins in yeast, structurally unrelated to Ric-8, are shown to be important for the biogenesis and signaling of Gα subunits and act as non-receptor GEFs (Lee and Dohlman, 2008Lee M.J. Dohlman H.G. Coactivation of G protein signaling by cell-surface receptors and an intracellular exchange factor.Curr. Biol. 2008; 18: 211-215Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). There are two isoforms of Ric-8 identified in vertebrates: Ric-8A, which acts within the biosynthetic pathway of the Gαi/o, Gαq/11, and Gα12/13 subclasses, and Ric-8B, which primarily acts on the Gαs/olf subfamily (Chan et al., 2013Chan P. Thomas C.J. Sprang S.R. Tall G.G. Molecular chaperoning function of Ric-8 is to fold nascent heterotrimeric G protein α subunits.Proc. Natl. Acad. Sci. USA. 2013; 110: 3794-3799Crossref PubMed Scopus (50) Google Scholar, Gabay et al., 2011Gabay M. Pinter M.E. Wright F.A. Chan P. Murphy A.J. Valenzuela D.M. Yancopoulos G.D. Tall G.G. Ric-8 proteins are molecular chaperones that direct nascent G protein α subunit membrane association.Sci. Signal. 2011; 4: ra79Crossref PubMed Scopus (73) Google Scholar, Nagai et al., 2010Nagai Y. Nishimura A. Tago K. Mizuno N. Itoh H. Ric-8B stabilizes the alpha subunit of stimulatory G protein by inhibiting its ubiquitination.J. Biol. Chem. 2010; 285: 11114-11120Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The isotypes Gαq/11, Gα12/13, and Gαolf appear to have the most stringent requirements for Ric-8 in order to be correctly folded and trafficked to the plasma membrane (Chan et al., 2011aChan P. Gabay M. Wright F.A. Kan W. Oner S.S. Lanier S.M. Smrcka A.V. Blumer J.B. Tall G.G. Purification of heterotrimeric G protein alpha subunits by GST-Ric-8 association: primary characterization of purified G alpha(olf).J. Biol. Chem. 2011; 286: 2625-2635Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, Gabay et al., 2011Gabay M. Pinter M.E. Wright F.A. Chan P. Murphy A.J. Valenzuela D.M. Yancopoulos G.D. Tall G.G. Ric-8 proteins are molecular chaperones that direct nascent G protein α subunit membrane association.Sci. Signal. 2011; 4: ra79Crossref PubMed Scopus (73) Google Scholar). Recent crystal structures of a truncated form of Ric-8A alone or in complex with a peptidomimetic of the C-terminal α5 helix of the G protein transducin (Gt) showed that the chaperone is composed of a combination of armadillo (ARM) and Huntington, Elongation Factor 3, PR65/A, TOR1 (HEAT) repeats. The ARM/HEAT repeats form a crescent-shaped core domain, with its concave surface engaging the α5 helix peptidomimetic (Srivastava et al., 2019Srivastava D. Gakhar L. Artemyev N.O. Structural underpinnings of Ric8A function as a G-protein α-subunit chaperone and guanine-nucleotide exchange factor.Nat. Commun. 2019; 10: 3084Crossref PubMed Scopus (10) Google Scholar, Zeng et al., 2019Zeng B. Mou T.C. Doukov T.I. Steiner A. Yu W. Papasergi-Scott M. Tall G.G. Hagn F. Sprang S.R. Structure, Function, and Dynamics of the Galpha Binding Domain of Ric-8A.Structure. 2019; 27: 1137-1147Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). However, the lack of structural information on full-length Ric-8-Gα complexes has hindered our understanding of the chaperone activity and the mechanism of Gα release from Ric-8, which presumably takes place upon successful Gα folding. Part of the challenge in attaining high-resolution information on folding intermediates is the intrinsic difficulties in obtaining stable complexes that are suitable for structural studies. In general, reported crystal structures of chaperone-client complexes lack high-resolution features of the client protein, thereby limiting the characterization of chaperoning mechanisms at large. To delineate the critical structural components of the chaperoning and GEF activity of Ric-8 toward Gα protein subunits, we sought to visualize these complexes by cryoEM. To this end, we purified or reconstituted native folding intermediates of two Gα subunits, Gαq and Gαi1, in complex with Ric-8A in the absence of guanine nucleotides. CryoEM maps of Ric-8A-Gαq and Ric-8A-Gαi1 at near-atomic resolution provide views of a unique chaperone-client complex and illustrate a striking chaperone mechanism that is employed by Ric-8A to stabilize critical elements for guanine nucleotide coordination by Gα. Mutagenesis coupled to both nucleotide exchange assays and a cellular chaperoning readout indicates that the recognition of the most C-terminal residue of Gα by Ric-8 provides a critical checkpoint for GTP binding to the G protein subunit and its subsequent release from the chaperone. Collectively, these results suggest a quality control mechanism underlying the chaperone and GEF activity of Ric-8 on Gα subunits. For our studies, we employed wild-type full-length Gαq or Gαi1 proteins and full-length Ric-8A, including its potentially unstructured regions, such as the Ric-8A C-terminal tail, which is required for chaperone activity (Oner et al., 2013Oner S.S. Maher E.M. Gabay M. Tall G.G. Blumer J.B. Lanier S.M. Regulation of the G-protein regulatory-Gαi signaling complex by nonreceptor guanine nucleotide exchange factors.J. Biol. Chem. 2013; 288: 3003-3015Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, Papasergi-Scott et al., 2018Papasergi-Scott M.M. Stoveken H.M. MacConnachie L. Chan P.Y. Gabay M. Wong D. Freeman R.S. Beg A.A. Tall G.G. Dual phosphorylation of Ric-8A enhances its ability to mediate G protein α subunit folding and to stimulate guanine nucleotide exchange.Sci. Signal. 2018; 11: eaap8113https://doi.org/10.1126/scisignal.aap8113Crossref PubMed Scopus (7) Google Scholar, Thomas et al., 2011Thomas C.J. Briknarová K. Hilmer J.K. Movahed N. Bothner B. Sumida J.P. Tall G.G. Sprang S.R. The nucleotide exchange factor Ric-8A is a chaperone for the conformationally dynamic nucleotide-free state of Gαi1.PLoS ONE. 2011; 6: e23197Crossref PubMed Scopus (42) Google Scholar). We chose to study these complexes without any stabilizing agent such as antibody fragments or crosslinkers, aiming to preserve both the native structure as well as the conformational flexibility between the chaperone and the client Gα subunit. It has been previously shown that site-specific phosphorylation of Ric-8A is critical for both its chaperone and GEF activity (Papasergi-Scott et al., 2018Papasergi-Scott M.M. Stoveken H.M. MacConnachie L. Chan P.Y. Gabay M. Wong D. Freeman R.S. Beg A.A. Tall G.G. Dual phosphorylation of Ric-8A enhances its ability to mediate G protein α subunit folding and to stimulate guanine nucleotide exchange.Sci. Signal. 2018; 11: eaap8113https://doi.org/10.1126/scisignal.aap8113Crossref PubMed Scopus (7) Google Scholar). To achieve these post-translational modifications, we utilized a baculovirus system to express Ric-8A in insect cells, a strategy that has been shown to produce largely phosphorylated chaperone at positions S435, T440, S522, S523, and S527 (Papasergi-Scott et al., 2018Papasergi-Scott M.M. Stoveken H.M. MacConnachie L. Chan P.Y. Gabay M. Wong D. Freeman R.S. Beg A.A. Tall G.G. Dual phosphorylation of Ric-8A enhances its ability to mediate G protein α subunit folding and to stimulate guanine nucleotide exchange.Sci. Signal. 2018; 11: eaap8113https://doi.org/10.1126/scisignal.aap8113Crossref PubMed Scopus (7) Google Scholar). In the case of Ric-8A-Gαi1, a complex stable enough for cryoEM imaging was generated by in vitro reconstitution from purified Ric-8A and myristoylated Gαi1 proteins, recombinantly expressed in insect cells and E. coli, respectively (Figure S1). Apyrase was added to hydrolyze the GDP released from Gαi1 upon forming a complex with Ric-8A in order to promote formation of a stable nucleotide-free complex. Due to the lower expression level and protein quality of Gαq compared to Gαi1 in the absence of chaperone and Gβγ, we isolated the Ric-8A-Gαq complex after co-expression of Ric-8A and Gα protein in insect cells. The complex was purified by utilizing an N-terminal GST tag on Ric-8A that was subsequently cleaved by TEV protease treatment. Prior to GST protein purification, apyrase was added to the lysed insect cell suspension for maximal stabilization of the Ric-8A-Gαq complex by hydrolyzing free guanine nucleotides. After GST purification and protease cleavage, the Ric-8A-Gαq complex was further purified by size-exclusion chromatography in order to separate it from free Ric-8A and GST protein. Both purification strategies resulted in stable complexes that were monodisperse as assessed by size-exclusion chromatography and negative stain EM visualization (Figure S1). From these samples, we obtained cryoEM maps of Ric-8A-Gαq and Ric-8A-Gαi1 with a global indicated resolution of 3.5 Å and 4.1 Å, respectively (Figure 1; Figures S1–S3, S4A, S4B, and Table S1). The structured portion of these assemblies is only ∼65 kDa, reinforcing the feasibility of conventional cryoEM for challenging proteins or complexes in the range of 50–100 kDa. These cryoEM maps enabled the modeling of Ric-8A between residues 1–482 in the Ric-8A-Gαi1 complex and residues 1–451 in the Ric-8A-Gαq complex, while the backbone of the C-terminal region (458–484) was traced with poly-alanine due to the poorer quality of the Ric-8A-Gαq map in the region 451–458. Nevertheless, both models included a relatively long stretch of the C-terminal region of Ric-8A (up to residue 482 or 484), which was not observed previously. The majority of the Ras-like domain residues of both Gαi1 (residues 32–54 and 193–354) and Gαq (residues 217–359) were also unambiguously modeled in these maps. Although the nucleotide binding regions of Gα are mostly ordered, we do not observe any density for a guanine nucleotide, consistent with the purification of both complexes in the presence of the nucleotide-hydrolyzing enzyme Apyrase and the known stability of Ric-8-Gα nucleotide-free complex. The absence of nucleotide destabilizes the closed conformation of Gα in which the α-helical domain is packed against the Ras-like domain. Thus, in both Ric-8A-Gαi1 and Ric-8A-Gαq complexes, the α-helical domain opens up and becomes flexible in relation to the rest of the complex, akin to its behavior in nucleotide-free G proteins in complex with a GPCR (Rasmussen et al., 2011Rasmussen S.G. DeVree B.T. Zou Y. Kruse A.C. Chung K.Y. Kobilka T.S. Thian F.S. Chae P.S. Pardon E. Calinski D. et al.Crystal structure of the β2 adrenergic receptor-Gs protein complex.Nature. 2011; 477: 549-555Crossref PubMed Scopus (2048) Google Scholar, Van Eps et al., 2015Van Eps N. Thomas C.J. Hubbell W.L. Sprang S.R. The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1.Proc. Natl. Acad. Sci. USA. 2015; 112: 1404-1409Crossref PubMed Scopus (17) Google Scholar, Westfield et al., 2011Westfield G.H. Rasmussen S.G. Su M. Dutta S. DeVree B.T. Chung K.Y. Calinski D. Velez-Ruiz G. Oleskie A.N. Pardon E. et al.Structural flexibility of the G alpha s alpha-helical domain in the beta2-adrenoceptor Gs complex.Proc. Natl. Acad. Sci. USA. 2011; 108: 16086-16091Crossref PubMed Scopus (168) Google Scholar). Accordingly, the general region occupied by the α-helical domain was masked out in our high-resolution map refinements for Ric-8A-Gαi1 and Ric-8A-Gαq complexes. However, in lower resolution unmasked reconstructions during initial steps of image processing, we observed partial density for the α-helical domain, jutted away from the nucleotide binding site in an average position that is rotated ∼90° compared to the nucleotide-bound structures of Gαi1 or Gαq (Figures 1E and 1F). Furthermore, the N-terminal helix of both G proteins is not resolved in the Ric-8A complexes, in agreement with its dynamic and unfolded state in the absence of Gβγ, as shown in previous crystal structures and by solution NMR studies of Gαi1 (Goricanec et al., 2016Goricanec D. Stehle R. Egloff P. Grigoriu S. Plückthun A. Wagner G. Hagn F. Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding.Proc. Natl. Acad. Sci. USA. 2016; 113: E3629-E3638Crossref PubMed Scopus (51) Google Scholar, Maly and Crowhurst, 2012Maly J. Crowhurst K.A. Expression, purification and preliminary NMR characterization of isotopically labeled wild-type human heterotrimeric G protein αi1.Protein Expr. Purif. 2012; 84: 255-264Crossref PubMed Scopus (5) Google Scholar, Medkova et al., 2002Medkova M. Preininger A.M. Yu N.J. Hubbell W.L. Hamm H.E. Conformational changes in the amino-terminal helix of the G protein alpha(i1) following dissociation from Gbetagamma subunit and activation.Biochemistry. 2002; 41: 9962-9972Crossref PubMed Scopus (55) Google Scholar). The core domain of Ric-8A (residues 1–421), comprised of a combination of nine ARM and HEAT repeats (R1–R9), adopts a crescent-shaped conformation that is similar to the one observed in recent crystal structures of truncated Ric-8A alone (Srivastava et al., 2019Srivastava D. Gakhar L. Artemyev N.O. Structural underpinnings of Ric8A function as a G-protein α-subunit chaperone and guanine-nucleotide exchange factor.Nat. Commun. 2019; 10: 3084Crossref PubMed Scopus (10) Google Scholar, Zeng et al., 2019Zeng B. Mou T.C. Doukov T.I. Steiner A. Yu W. Papasergi-Scott M. Tall G.G. Hagn F. Sprang S.R. Structure, Function, and Dynamics of the Galpha Binding Domain of Ric-8A.Structure. 2019; 27: 1137-1147Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar) (Figure S4C) or in complex with the Gαt α5 helix peptidomimetic (Srivastava et al., 2019Srivastava D. Gakhar L. Artemyev N.O. Structural underpinnings of Ric8A function as a G-protein α-subunit chaperone and guanine-nucleotide exchange factor.Nat. Commun. 2019; 10: 3084Crossref PubMed Scopus (10) Google Scholar) (Figure S4D), with an RMSD of 0.9–1 Å. This observation suggests that the Ric-8A core domain maintains an overall stable conformation in the presence and absence of Gα. Interestingly, however, the core domain of Ric-8A adopts a slightly more compact configuration in its complex with Gαq compared to Gαi1 (Figure S4B). Furthermore, the bulk of the observable Ric-8A C terminus (residues 421-482) becomes ordered in the presence of Gαi1 or Gαq. This region largely maintains a coil structure, which encapsulates the Ras-like domain of Gαq or Gαi1, before forming an α-helix most likely comprised of residues 474–484 for Gαq and 474–481 for Gαi1 (Figure 2). The last C-terminal 40 residues of Ric-8A (490–530), predicted to include a coil/β-strand and an α-helix/coil structure (Figure S4E), have been shown to be critical for the chaperone function of Ric-8A in vivo, but are not required for in vitro GEF activity (Oner et al., 2013Oner S.S. Maher E.M. Gabay M. Tall G.G. Blumer J.B. Lanier S.M. Regulation of the G-protein regulatory-Gαi signaling complex by nonreceptor guanine nucleotide exchange factors.J. Biol. Chem. 2013; 288: 3003-3015Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, Papasergi-Scott et al., 2018Papasergi-Scott M.M. Stoveken H.M. MacConnachie L. Chan P.Y. Gabay M. Wong D. Freeman R.S. Beg A.A. Tall G.G. Dual phosphorylation of Ric-8A enhances its ability to mediate G protein α subunit folding and to stimulate guanine nucleotide exchange.Sci. Signal. 2018; 11: eaap8113https://doi.org/10.1126/scisignal.aap8113Crossref PubMed Scopus (7) Google Scholar, Thomas et al., 2011Thomas C.J. Briknarová K. Hilmer J.K. Movahed N. Bothner B. Sumida J.P. Tall G.G. Sprang S.R. The nucleotide exchange factor Ric-8A is a chaperone for the conformationally dynamic nucleotide-free state of Gαi1.PLoS ONE. 2011; 6: e23197Crossref PubMed Scopus (42) Google Scholar). In our maps, this region appears disordered yet lies within the vicinity of the α-helical domain of Gα, raising the possibility that the 40 C-terminal residues may engage the flexible α-helical domain. The cryoEM structures of Ric-8A-Gαi1 and Ric-8A-Gαq reveal three primary interaction surfaces between the chaperone and Gα Ras-like domain (Figure 2A). One interface involves an extensive interaction of the C-terminal α5-helix of Gα with the concave surface of the ARM/HEAT repeats (Figure 2B). A second interface is formed by the β4, β5, and β6 strands of Gα with the R8-R9 ARM/HEAT repeats and a loop formed by residues 453–459 of the extended C terminus of Ric-8A (Figure 2C). The third interface is formed by the C-terminal helix of Ric-8A, which is in contact with switch2, the P loop, and the α3 helix of the Gα Ras-like domain (Figure 2D). A comparison of the Gα Ras-like domain conformation in the Ric-8A-Gα maps and nucleotide-bound Gα crystal structures reveals several notable differences. The most distinct difference is the position of the α5 helix and the very C-terminal residues of Gαi1 and Gαq, which interact with the inner concave surface of the ARM/HEAT core domain of Ric-8A. In both complexes, the α5 helix is completely detached from the Ras-like domain and rotated by more than 90° away from its position in the Gα nucleotide-bound conformation (Figures 1E and 1F). The α5 helix is connected to the β6 strand by a loop containing the conserved TCAT motif, a critical element with residues that coordinate the guanine base of GDP or GTP bound to the Ras-like domain. Because of the displacement of the α5 helix (Figure 3A), the TCAT is positioned 3–5 Å away from its nucleotide coordination site (Figure 3B). The positioning of the TCAT motif of Gαi1 and Gαq in complex with Ric-8A is similar to its configuration in nucleotide-free heterotrimeric Gαi1-Gβγ and Gα11-Gβγ (Gα11 is a close homolog of Gαq) proteins in complex with GPCRs (Figures S5A and S5B). Of note, the displacement of the α5 helix from the Ras-like domain core may be promoted by the second helix of the final HEAT repeat (αB9) of Ric-8. Structural alignment of the Ras-like domains shows that the αB9 helix in the Ric-8A-bound Gαi1 structure occupies the position of the α5 helix in the GTPγS-bound Gαi1 structure (Figures S5C and S5D). Another distinct difference in the Ras-like domain involves the conformation of the switch2 motif and α2 helix that are also important for Gα nucleotide binding. The switch2/α2 elements are mostly disordered in the Ric-8A-Gαi1 structure, while in the map of the Ric-8A-Gαi1 complex, we can trace their density away from their corresponding conformations in the Gα GTP-bound state (Figures 3A and 3B). This positioning is induced by a C-terminal helix of Ric-8A (474–484), which is sandwiched between the switch2 motif and α3 helix, potentially forming interactions with the P loop and α2 and α3 helices. The switch1 motif of Gα appears disordered in both of our Ric-8A complex structures, potentially due to the conformational flexibility of the α-helical domain. Additionally, the β2 and β3 strands together with α1 helix of the Ras-like domain are partially ordered in the Ric-8A-Gαq structure and modestly more ordered in the Ric-8A-Gαi1 structure. Notably, the map densities corresponding to these secondary structure elements in both the Gαi1 and Gαq complexes consistently displayed variability across several 3D classes. Although this region is too limi" @default.
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• W3010123279 date "2020-03-01" @default.
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• W3010123279 title "Structures of Gα Proteins in Complex with Their Chaperone Reveal Quality Control Mechanisms" @default.
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