Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit

Abstract

In G-protein signaling, an activated receptor catalyzes GDP/GTP exchange on the G α subunit of a heterotrimeric G protein. In an initial step, receptor interaction with G α acts to allosterically trigger GDP release from a binding site located between the nucleotide binding domain and a helical domain, but the molecular mechanism is unknown. In this study, site-directed spin labeling and double electron-electron resonance spectroscopy are employed to reveal a large-scale separation of the domains that provides a direct pathway for nucleotide escape. Cross-linking studies show that the domain separation is required for receptor enhancement of nucleotide exchange rates. The interdomain opening is coupled to receptor binding via the C-terminal helix of G α , the extension of which is a high-affinity receptor binding element.signal transduction | structural polymorphism T he α-subunit (G α ) of heterotrimeric G proteins (G αβγ ) mediates signal transduction in a variety of cell signaling pathways (1). Multiple conformational states of G α are involved in the signal transduction pathway shown in Fig. 1A. In the inactive state, the G α subunit contains a bound GDP [G α ðGDPÞ] and has a high affinity for G βγ . When activated by an appropriate signal, a membrane-bound G-protein coupled receptor (GPCR) binds the heterotrimer in a quaternary complex, leading to the dissociation of GDP and formation of an "empty complex" [G α ð0Þ βγ ], which subsequently binds GTP. The affinity of G α ðGTPÞ for G βγ is dramatically reduced relative to G α ðGDPÞ, resulting in functional dissociation of active G α ðGTPÞ from the membrane-bound complex. The active G α ðGTPÞ subsequently binds downstream effector proteins to trigger a variety of regulatory events, depending on the particular system. Thus, the GPCR acts to catalyze GDP/GTP exchange via an empty complex. Crystallographic (2-7), biochemical (8), and biophysical (9-11) studies have elucidated details of the conformational states of G α that correspond to the discrete steps indicated in Fig. 1A, but the mechanism by which receptor interaction leads to release of the bound GDP from G α and the structure of the empty complex remain a major target of research in the field.The G α subunit has two structural domains, namely a nucleotide binding domain and a helical domain that partially occludes the bound nucleotide (Fig. 1B). From the initial G α crystal structure in 1993, Noel et al. (2) recognized that nucleotide release would probably require an opening between the two domains in the empty complex, but in the intervening 18 years there has been little compelling experimental support for this idea. Nevertheless, some constraints on the general topology of the complex are known. For example, numerous studies indicate that the C terminus of G α is bound tightly to the receptor in the empty complex (9). In addition, the N-terminal helix of G α is associated with G βγ and with the membrane via N-terminal myristoylation (12,13). Together, these constraints fix the position of the nucleot...

The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

“…S2) and, thus, must be substantially longer then would be expected for a Gs:β2R-like complex (∼50 Å) in which the distance would be resolved. To the extent that they can be compared, the displacement and broad distribution in position of the Giα1 helical domain in the Ric-8A complex is similar to that observed in the complex with activated rhodopsin (20). A distribution of helical domain positions is also observed in cryo-EM studies of the β2R:Gs complex (40).…”

“…Thus, both GEFs bind to the C terminus (4,25,(36)(37)(38)(39) of Gα and trigger changes that destabilize the contacts between the helical and Ras-like domains of Gα. In addition to the appearance of an open state in which residue 90R1 in the helical domain is separated by ∼48 Å from 214R1 in switch II and 238R1 in switch III, there appears to be essentially a continuum of intermediate states that approach the fully closed Gαi1•GDP conformation, suggesting a ratchet-like separation that may facilitate nucleotide release while clearly providing an entry route to GTP (20,25) (Fig. 4A).…”

“…This vector was used for construction of double-cysteine mutants by QuikChange mutagenesis (Agilent). The C147/C235 mutant was constructed as described (20). Hexa I Gαi1 harboring cysteine pairs were expressed and purified as described by Thomas et al (4).…”

“…The data reveal that binding of Ric-8A to Gαi1 induces structural heterogeneity due to new conformations in which the helical domain has pivoted away from the Ras-like domain, exposing the nucleotide-binding site to solvent, thus providing an escape (and entry) pathway for the nucleotide. A similar change is induced in Gαi1 upon formation of the nucleotide-free complex with the activated GPCR rhodopsin (20), but is distinctly Significance Heterotrimeric G proteins are activated by exchange of a bound GDP for GTP at the active site of the alpha subunit (Gα), typically mediated by a transmembrane G protein-coupled receptor (GPCR). Resistance to inhibitors of cholinesterase 8A (Ric-8A), a soluble intracellular protein that is essential for embryonic development, can also catalyze nucleotide exchange and activate G proteins.…”

“…Inserted into switch I of the Ras domain is a helical domain that is unique to the family of heterotrimeric G proteins. The helical domain flanks the guanine nucleotide-binding site and, while it makes few direct contacts with the nucleotide, shields it from solvent and may affect the rate of its dissociation (20,21).…”