Snapshots of the dynamic basis of NTSR1 G protein subtype promiscuity

G-protein-coupled receptors (GPCRs) are capable of signalling through four families of G protein α subunits. Although hundreds of nucleotide-free GPCR-G protein complex structures have been solved, the mechanism of G protein subtype selectivity remains poorly understood, with recent studies suggesting a role for dynamic nucleotide-bound intermediate states^1,2. Here we use time-resolved cryo-electron microscopy to visualize the GTP-induced activation of Gα_i1βγ and Gα₁₁βγ heterotrimers bound to the neurotensin receptor 1 (NTSR1), which has been demonstrated to be highly promiscuous in G protein coupling and to possess unusual conformations in the nucleotide-free complex. We resolve ensembles of states along the G protein activation pathway, with differences in the structures and their relative populations between Gα_i1 and Gα₁₁. Structural analysis reveals a key role for several motifs, including intracellular loop 2 (ICL2) and ICL3, in stabilizing the observed intermediate states. Our results are supported by molecular dynamics simulations and kinetic bioluminescence resonance energy transfer experiments, which reveal that the stability of these intermediate states and the signalling of various G proteins are correlated with ICL2 and ICL3 sequences. Single-molecule fluorescence assays of GTP-induced NTSR1-G protein complex dissociation reveal that NTSR1 is liberated significantly faster from Gα₁₁, consistent with the relative lack of stable Gα₁₁-GTP intermediate states compared with Gα_i1. These findings highlight that transient intermediate-state complexes along the G protein activation pathway have an important role in G protein selection that cannot be explained by nucleotide-free states alone.