Guanine nucleotide binding proteins popularly known as G-proteins, involved in a variety of cellular signal transduction pathways are heterotrimeric proteins consisting of α, β, and γ subunits. Ric8A is known to be both a chaperone for the assembly of the α-subunit of G-proteins, and a Guanine nucleotide Exchange Factor (GEF). McClelland et al., have conducted a detailed structural analysis on the complex between Ric8A and Gαi1 using cryoEM, X-ray crystallography, and SAXS. Constructs of Ric8A and Gαi1 optimized for structure determination and to reduce conformational heterogeneity were used to assemble the Ric8A-Gαi1 complex. They were able to determine the interface between the two proteins which consists of three separate surface contacts which essentially stabilize Gα in its nucleotide-free state. Furthermore, it was found that a specific Casein Kinase mediated phosphorylation of Ric8A stimulated the GEF activity by structurally stabilizing the Ric8A-Gα interface. Ric8A binding to the disrupted Guanine nucleotide binding site on Gα was determined to be critical for the GEF function as point mutants at residues of Ric8A involved in this interaction compromised the GEF activity. A significant amount of re-organization of a particular helical domain in Gα relative to the GTPase domain is involved in providing a means for the nucleotide to exit. The magnitude of this re-orientation is underrepresented in the crystal and EM structures possibly due to the steric hindrance inflicted by the nano-bodies used as a strategy to minimize flexibility in the structure. SAXS data acquired on the complex without the nano-bodies provided a picture of the real extent of conformational variation thus illustrating the advantage of using multiple complementary approaches to address any given structural biological question.
See: McClelland LJ, Zhang K, Mou TC, Johnston J, Yates-Hansen C, Li S, Thomas CJ, Doukov TI, Triest S, Wohlkonig A, Tall GG, Steyaert J, Chiu W, Sprang SR. “Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1”. Nat Commun. 2020 Feb 26;11(1):1077. DOI: 10.1038/s41467-020-14943-4. PMID:32103024