Introduction. In recent years there has been an exponential growth in the amount of solved inactive G protein-coupled receptor (GPCR) structures, however there still remains a dearth of active-state, G-protein-bound, GPCRs. To date, all solved active-state GPCRs have been coupled to the stimulatory Gs protein. The class A adenosine A1 receptor (A1R) is a GPCR that preferentially couples to the inhibitory Gi/o family of heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted.
Aims. We sought to solve an active-state structure of the A1R bound to the Gαi2 inhibitory G protein.
Methods. Generation of a dominant negative Gαi2 enabled formation of a stabilized ternary complex of the adenosine bound A1R with Gi2, which was purified to homogeneity and subjected to Volta phase plate cryo-electron microscopy.
Results. We were successful in solving a 3.6 Å structure of the human A1R-Gi2 ternary complex. Compared to the inactive A1R, there is contraction at the extracellular surface upon activation in the wide orthosteric binding site that is mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A1R primarily via amino acids in the C-terminus of the Gɑi ɑ5 helix, concomitant with a 10.5 Å outward movement of A1R transmembrane domain 6. Comparison with the agonist-bound β2 adrenergic receptor·Gs-protein complex reveals distinct orientations for each G protein subtype upon engagement with its receptor.
Discussion. This active A1R structure provides novel molecular insights into receptor/G protein selectivity.