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G protein-coupled receptors represent the largest family of membrane receptors1 that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human β2 adrenergic receptor (β2AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the β2AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the β2AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the 'P-loop' that binds the β-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity.
The formation of a complex between a G protein-coupled receptor (GPCR) and a heterotrimeric G protein is responsible for the majority of transmembrane signalling in response to hormones and neurotransmitters. Heterotrimeric G proteins are composed of a nucleotidebinding α-subunit (Ga) and an obligate dimer of the b and c-subunits (Gβγ). In their inactive form, Gα-subunits are bound to GDP and tightly associated with Gβγ. Gα proteins are evolutionarily related to the Ras family of G proteins but contain a small globular domain, referred to as the α-helical domain (GαsAH) (Fig. 1a). The Ras-like GTPase domain (GasRas) contains most of the catalytic residues necessary for GTP hydrolysis, as well as the Gβγ and effector binding regions4. The structures within these regions differ between GTP- and GDP-bound states, and are appropriately termed switch regions5,6.
The process ofG protein activation involves formation of a complex (often referred to as a ternary complex)...