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

Abstract

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.DOI: http://dx.doi.org/10.7554/eLife.19238.001

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

“…In contrast, segments that directly surround the nucleotide‐binding site, the P‐loop, and the guanine base interacting β5‐αG loop/αG region revealed increased rates of HDX exchange indicative of a protein backbone destabilization. The observed deprotection extended to the scaffolding β1, β4, and β5 strands not previously reported for the G‐protein/GPCR complexes . Thus, the interface of Gα with Ric‐8 appears to be more extensive than that with GPCRs, and it incorporates sites utilized by GPCRs as well as by GBA‐motif proteins (the switch II region).…”

“…In addition, the αN‐β1 loop, the β1–β3 strands, and to a lesser extent, the switch II region wrap around portions of the proximal C‐terminal tail of Ric‐8A (Figures 5 and 6). Hence, the model is consistent with destabilization of the Gα scaffolding β‐sheet observed in the complex with Ric‐8A …”

“…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 .…”

“…However, while Ric‐8A1‐452 retains substantial capacity to catalyze GDP release, its GEF activity appears to be limited by the rate of GTPγS binding . Binding of GTPγS to a preformed nucleotide‐free complex of Gα with Ric‐8A1‐452 was shown to be strongly impaired as well . From the Ric‐8A1‐452/Gα model we infer that disorganization of the Gα nucleotide‐binding site in the Ric‐8A/Gα complex is so significant, that unless Ric‐8A453‐492 assists the binding of GTP, the latter reaction and GαGTP release from the complex limit the GEF activity of Ric‐8A.…”

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

“…Biophysical data show that nucleotide-free Gαi1 is structurally dynamic when bound to Ric-8A 14, 15 and shares some properties with GPCR-bound Gα 16 . Namely, Ric-8A induces rotational dynamics in the Gα helical domain 17 and binds to the C-terminus of Gα 14, 18 .…”