Effect of α-helical domain of Gi/o α subunit on GDP/GTP turnover

Abstract

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of α, β, and γ subunits, and Gα has a GDP/GTP-binding pocket. When a guanine nucleotide exchange factor (GEF) interacts with Gα, GDP is released, and GTP interacts to Gα. The GTP-bound activated Gα dissociates from GEF and Gβγ, mediating the induction of various intracellular signaling pathways. Depending on the sequence similarity and cellular function, Gα subunits are subcategorized into four subfamilies: Gαi/o, Gαs, Gαq/11, and Gα12/13. Although the Gαi/o subtype family proteins, Gαi3 and GαoA, share similar sequences and functions, they differ in their GDP/GTP turnover profiles, with GαoA possessing faster rates than Gαi3. The structural factors responsible for these differences remain unknown. In this study, we employed hydrogen/deuterium exchange mass spectrometry and mutational studies to investigate the factors responsible for these functional differences. The Gα subunit consists of a Ras-like domain (RD) and an α-helical domain (AHD). The RD has GTPase activity and receptor-binding and effector-binding regions; however, the function of the AHD has not yet been extensively studied. In this study, the chimeric construct containing the RD of Gαi3 and the AHD of GαoA showed a GDP/GTP turnover profile similar to that of GαoA, suggesting that the AHD is the major regulator of the GDP/GTP turnover profile. Additionally, site-directed mutagenesis revealed the importance of the N-terminal part of αA and αA/αB loops in the AHD for the GDP/GTP exchange. These results suggest that the AHD regulates the nucleotide exchange rate within the Gα subfamily.

Structural and Functional Implication of Natural Variants of Gαs

“…RD is critical for canonical Gα functions, such as intrinsic GTPase activity and interaction with Gβγ ( Figure 1 A), receptors ( Figure 1 B), and downstream signaling proteins ( Figure 1 D) [ 1 , 10 , 11 , 13 ]. The functions of AHD have not been studied extensively, but it has been described as the regulator of the GDP/GTP turnover rate [ 46 , 47 , 48 ].…”

“…We investigated whether these natural variants mutated in Gαs AHD affect the intrinsic GDP/GTP turnover rate using BODIPY-FL-GTPγS. BODIPY-FL-GTPγS has been successfully used to measure the relative GDP/GTP turnover rate [ 48 , 49 ]. The BODIPY fluorescence intensity of BODIPY-FL-GTPγS in solution is quenched by the neighboring guanine ring of GTPγS owing to the flexible bending of the linker.…”

“…A previous study reported that the conformational dynamics of AHD could affect the GDP/GTP turnover rate. Dohlman et al revealed the X-ray crystal structure of GPA1, a self-activating G protein α subunit of Arabidopsis thaliana , which suggested that the higher conformational fluctuation at αA of GPA1 than that of Gαi1 is related to the faster GDP/GTP turnover rate of GPA1 than of Gαi1 [ 46 , 47 , 48 ]. In a recent study, we reported that AHD is responsible for the different GDP/GTP turnover rates between Gαi and Gαo subtypes [ 51 ].…”

Stabilization of Interdomain Interactions in G protein αiSubunits Determines GαiSubtype Signaling Specificity

“…RLD-HD interactions have classically been understood to be a regulator of nucleotide exchange 12,[40][41][42][43][44][45] , with mutations at the interface intended to disrupt interactions leading to higher rates of GDP dissociation 12 . Specifically, mutation of residue R144 in Gα i1 to an alanine is known to significantly increase the rate of GTPγS binding, presumably through the breaking of an interdomain interaction with L232 12 .…”