Heterotrimeric G proteins (G proteins) are pivotal regulators of cellular signaling pathways and operate downstream of G protein-coupled receptors (GPCRs). Composed of three subunits—Gα, Gβ, and Gγ—the activation of G proteins is tightly regulated by the nucleotide-binding state of the Gα subunit, specifically the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) (Milligan and Kostenis, 2006). In the inactive state, Gα forms a heterotrimeric complex with Gβγ. Upon activation by GPCRs, GDP is released from Gα, and the abundant cytosolic GTP (Traut, 1994) rapidly binds to the nucleotide-free Gα. This nucleotide exchange induces conformational changes in Gα, leading to its dissociation from Gβγ and activation of downstream effectors such as adenylate cyclase or phospholipase C (Gilman, 1984, Northup et al., 1982, Northup et al., 1980, Smrcka et al., 1991, Sternweis and Smrcka, 1992, Taylor et al., 1991). The intrinsic GTPase activity of Gα hydrolyzes GTP back to GDP, resetting the protein to its basal state and enabling reassociation with Gβγ (Milligan and Kostenis, 2006). Based on their Gα subunits, G proteins are classified into four subfamilies: Gs, Gi/o, Gq/11, and G12/13.

Since the high-resolution structure of the β2-adrenergic receptor (β2AR)-Gs complex was elucidated in 2011 (Fig. 1A), numerous GPCR-G protein complexes with diverse coupling partners have been structurally characterized, as summarized in GPCRdb.org. These structural studies have revealed critical insights into the interaction interfaces between GPCRs and G proteins and the allosteric conformational changes involved in their coupling (Glukhova et al., 2018, Hilger et al., 2018). Among these interaction sites, the C-terminal α5 helix of Gα, which engages the cytosolic core of the receptor (Fig. 1A), has emerged as a key determinant of GPCR-G protein complex formation and coupling selectivity (Du et al., 2019, Inoue et al., 2019, Liu et al., 2019b, Sandhu et al., 2019). Particularly, the C-terminal 5 residues of Gα (commonly referred to as the “wavy hook”) (Fig. 1A, green sticks in the enlarged structure) have been considered to be crucial for these functions. Our prior work, using wavy hook-truncated Gαs and Gαi constructs, has suggested that this region serves as the initial contact site for GPCR-G protein coupling and plays a pivotal role in coupling specificity (Du et al., 2019, Kim et al., 2020).

Although previous studies have emphasized the structural and functional importance of the wavy hook in GPCR coupling, its potential role in regulating Gα’s intrinsic GDP/GTP turnover, GTPase activity, and conformational dynamics remains unexplored. In this study, we deleted the wavy hook (the C-terminal five residues: DCGLF for Gαi1 and QYELL for Gαs) and assessed its effects on these intrinsic properties using fluorescence-based BODIPY assays and hydrogen/deuterium exchange mass spectrometry (HDX-MS). Our findings provide insight into the subtype-specific role of the wavy hook in stabilizing G protein structure and function, with implications for understanding GPCR signaling mechanisms.