Structure, Function, and Dynamics of the Gα Binding Domain of Ric-8A

Zeng, Baisen; Mou, Tung-Chung; Doukov, Tzanko; Steiner, Andrea; Yu, Wenxi; Papasergi-Scott, Makaía M.; Tall, Gregory G.; Hagn, Franz; Sprang, Stephen R.

doi:10.1016/j.str.2019.04.013

Cited by 17 publications

(58 citation statements)

References 63 publications

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“…N-terminally glutathione-S-transferase tagged Rat Gαi1 with a 31 residue N-terminal truncation (ΔN31Gαi1) was expressed from a pDest15 vector in Bl21(DE3) RIPL Escherichia coli and purified as previously described 1, 9 . N-terminally hexahistidine tagged Rat Ric-8A(residues 1-491, hereafter Ric-8A) was expressed and purified as previously described in a pET28a vector with BL21(DE3) RIPL Escherichia coli cells 1, 3, 10 . Following removal of the hexahistidine tag using Tobacco Etch Virus protease, Ric-8A protein was loaded onto a Source 15Q column and eluted with a 500 mM NaCl gradient at 180 mM NaCl and subsequently loaded on a GE Healthcare HiLoad 16/600 Superdex 200 column for size exclusion chromatography in gel filtration buffer containing 50 mM Tris pH 8, 150 mM NaCl and 1 mM Tris(2-carboxyethyl)phosphine (TCEP).…”

Section: Methodsmentioning

confidence: 99%

“…Ric-8A was phosphorylated using casein kinase II (New England Biolabs) as previously described 10, 11 . Briefly, purified Ric-8A was mixed 1:1 with a 2X Kinase Reaction Buffer containing 0.1 M HEPES pH 8, 0.1 M NaCl, 20 mM MgCl 2 , 2 mM EGTA, and 1 mM DTT.…”

Section: Methodsmentioning

confidence: 99%

“…All procedures described hereinafter were conducted with phosphorylated Ric-8A comprising residues 1-491 of the full-length protein and referred to as Ric-8A. Myristoylated Gαi1 (mGαi1), used in nucleotide exchange assays, was prepared as described previously 10 . Mutants of Ric-8A, used in nucleotide exchange assays, were constructed via the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent).…”

Section: Methodsmentioning

confidence: 99%

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Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

McClelland

Zhang

Mou

et al. 2020

Preprint

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23 USA 24 † Present address, Regeneron Pharmaceutical, Inc. USA 25 26 Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates 27 heterotrimeric G protein alpha subunits (Gα) 1 . Ric-8A is essential to life in 28 multicellular eukaryotes by virtue of its chaperone activity that is required for Gα 29 biogenesis and membrane localization 2,3 . Ric-8A adopts an armadillo (ARM)/HEAT 30 repeat domain architecture and is structurally unrelated to G Protein-Coupled 31 Receptors (GPCR) 4 . Both GEF and chaperone activities are stimulated by Casein 32 Kinase II phosphorylation 5 . The mechanisms by which Ric-8A catalyzes GDP 33 release and GTP binding to Gα, or exerts chaperone activity are unknown. Here, 92 Table 2). Initial crystallographic phases were determined by molecular replacement 93 and subsequently used to fit the cryo-EM density map. Iterative cycles of model-building 94 and refinement utilized both cryo-EM and X-ray diffraction data to generate the final 95 models (Extended Data Table S1 and S2). In the following discussion, we use 96 prefixes "r" and "g" for residue and secondary structure identifiers of Ric-8A and Gα, 97 respectively. ARM/HEAT repeat helices of Ric-8A are designated according to their 98 position in the repeat: "A", "B" or "C" for ARM repeats or "a" and "b" for HEAT repeats, 99 followed by the sequence number of the repeat (1 through 9). We use the established 100 nomenclature for Gα secondary structure [21][22][23] . Descriptions of the crystal structure of 101 Ric-8A:Gα refer to chains A and B, the better ordered of the two complexes in the 102 asymmetric unit. 103The cryo-EM reconstruction reveals Ric-8A residues 2-487 and the whole of Gα with 104 the exception of the disordered linker (residues 50-76) between the Helical and GTPase 105 domains ( Fig. 1a and 1b and Extended Data Video 1). The crystal structure of the Ric- 1068A:Gα complex also reveals continuous electron density for Ric-8A except for the 107 linkage between 422 and 430. Notably, residues C-terminal to the last HEAT repeat 108 (r430-r491) are disordered in structures of Ric-8A in which Gα is absent 4,18 . In the 109 crystal structure, which lacks Nb1056, the helical domain of Gαi1 and its connections to 110 the GTPase domain, including much of gα1 and all of switch I, are disordered. 111Otherwise the cryo-EM and X-ray models of Ric-8A:Gα are in good agreement, although 112 certain irregularly-structured and loop regions in both Gα and Ric-8A show significant 113 divergence (Extended Data Fig. 6). Small angle X-ray scattering (SAXS) 114 measurements of the complex are consistent with the X-ray and cryo-EM structures 115 (Extended Data Fig. 7).116 Figure 2. Interactions of Ric-8A with Gαi1. Three major Ric-8A:Gα contact surfaces and phosphorylation 117 sites are highlighted and labeled in the green, yellow, pink and blue overlays (upper left) and enlarged in 118 panels a-d. Axes adjacent to each panel label indicate the rotation applied to the overall schematic to 119 generate the panel view. a, Interac...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

McClelland

Zhang

Mou

et al. 2020

Preprint

Self Cite

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“…For a long period of time, Ric‐8A escaped attempts to determine its structure by means of X‐ray crystallography. A probable reason for the “reluctance” of Ric‐8 to form well‐diffracting crystals became apparent following the recent solution of its structure . The structure of the active nearly full‐length Ric‐8A1‐492 revealed two principal domains of Ric‐8A: an armadillo‐like core (residues about 1–426) and an unstructured C‐terminal tail (residues 427–492) .…”

Section: Biochemical and Structural Advances Toward Understanding Thementioning

confidence: 99%

“…Indeed, the distal C‐tail is important for the GEF activity of Ric‐8A. Various degrees of impairment of the GEF activity have been reported for the truncated Ric‐8A1‐452 construct, but a preponderance of the data suggests that it is a very poor GEF compared to the full‐length Ric‐8A or Ric‐8A1‐492 . Moreover, a potential Gα‐binding site has been proposed for residues Ric‐8A455‐470 .…”

Section: Biochemical and Structural Advances Toward Understanding Thementioning

confidence: 99%

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

Srivastava

Artemyev

2020

BioEssays

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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.

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Activators of G ‐Protein Signaling in the Normal and Diseased Kidney

Park

2022

GPCRs as Therapeutic Targets

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Structure, Function, and Dynamics of the Gα Binding Domain of Ric-8A

Cited by 17 publications

References 63 publications

Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

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

Activators of G ‐Protein Signaling in the Normal and Diseased Kidney

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