Structural flexibility of the Gas a-helical domain in the β_2-adrenoceptor Gs complex

摘要

The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotrans-mitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied β_2-adrenoceptor (β_2AR) in complex with the heterotrimeric G protein Gs (Gαsβ_γ). EM 2D averages and 3D reconstructions of the deter-gent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the a-helical domain of Gas appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphono-formate), and also the presence of GDP, favor the stabilization of the a-helical domain on the Ras-like domain of Gas. Molecular modeling of the a-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of Gas-GTPγS. These data argue that the a-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.