Conformational changes in the G protein Gs induced by the beta(2) adrenergic receptor

Abstract

G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein ?-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 ?2 adrenergic receptor (?2AR). 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 ?2AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the ?2AR-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 ?-subunit of Gs and consequently alters the 'P-loop' that binds the ?-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and ?-phosphate coordination are key determinants of GDP (and GTP) binding affinity. [ABSTRACT FROM AUTHOR]