The structural basis of the dominant negative phenotype of the Gαi1β1γ2 G203A/A326S heterotrimer

Abstract

Aim:Dominant negative mutant G proteins have provided critical insight into the mechanisms of G protein-coupled receptor (GPCR) signaling, but the mechanisms underlying the dominant negative characteristics are not completely understood. The aim of this study was to determine the structure of the dominant negative Gαi1β1γ2 G203A/A326S complex (Gi-DN) and to reveal the structural basis of the mutation-induced phenotype of Gαi1β1γ2.Methods:The three subunits of the Gi-DN complex were co-expressed with a baculovirus expression system. The Gi-DN heterotrimer was purified, and the structure of its complex with GDP was determined through X-ray crystallography.Results:The Gi-DN heterotrimer structure revealed a dual mechanism underlying the dominant negative characteristics. The mutations weakened the hydrogen bonding network between GDP/GTP and the binding pocket residues, and increased the interactions in the Gα-Gβγ interface. Concomitantly, the Gi-DN heterotrimer adopted a conformation, in which the C-terminus of Gαi and the N-termini of both the Gβ and Gγ subunits were more similar to the GPCR-bound state compared with the wild type complex. From these structural observations, two additional mutations (T48F and D272F) were designed that completely abolish the GDP binding of the Gi-DN heterotrimer.Conclusion:Overall, the results suggest that the mutations impede guanine nucleotide binding and Gα-Gβγ protein dissociation and favor the formation of the G protein/GPCR complex, thus blocking signal propagation. In addition, the structure provides a rationale for the design of other mutations that cause dominant negative effects in the G protein, as exemplified by the T48F and D272F mutations.

Structure of formylpeptide receptor 2-Gi complex reveals insights into ligand recognition and signaling

“…1A and B). We used human Gαi1 with two dominant negative mutations 29 , rat Gβ1 and bovine Gγ2 to form the Gi heterotrimer. We added an antibody fragment scFv16 to stabilize the nucleotide-free complex by binding to the interface between Gα i and Gβ 30 ( Supplementary Fig.…”

“…The prolactin precursor sequence was inserted into the N terminal to increase the protein expression. A dominant-negative bovine Gαi1 (DNGαi1) construct was generated by site-directed mutagenesis to incorporate mutations G203A and A326S to decrease the affinity of nucleotide binding and increase the stability of Gαβγ complex 29 . All the three G protein complex components, DNGαi1, rat Gβ1 and bovine Gγ2, were cloned into pFastbac individually.…”

C10ORF12 modulates PRC2 histone methyltransferase activity and H3K27me3 levels

“…A 10 μL sample with a protein concentration of 1 mg/mL was loaded on a Nanofilm SEC-500 (Sepax) column in size buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl) with a flow rate set at 0.35 mL/min. The molecular weight of the complex was then calculated on the basis of the differential refractive index signal determined by the intensity of light scattering and the coinciding protein concentration [54].…”

Molecular mechanisms of fentanyl mediated β-arrestin biased signaling

“…High-titer recombinant baculovirus (>10 9 virus particles per ml) of μOR (wild type and mutants), Gαi1, Gβ1 and Gγ2 was obtained using Bac-to-Bac Baculovirus Expression System (Invitrogen) as previously described [57,58]. Cell suspensions were cultured for 4 days while shaking at 27˚C to generate P1 virus.…”