Probing Gαi1 protein activation at single-amino acid resolution

We present comprehensive maps at single-amino acid resolution of the residues stabilizing the human G alpha(i1) subunit in nucleotide- and receptor-bound states. We generated these maps by measuring the effects of alanine mutations on the stability of G alpha(i1) and the rhodopsin-G alpha(i1) complex. We identified stabilization clusters in the GTPase and helical domains responsible for structural integrity and the conformational changes associated with activation. In activation cluster I, helices alpha 1 and alpha 5 pack against strands beta 1-beta 3 to stabilize the nucleotide-bound states. In the receptor-bound state, these interactions are replaced by interactions between alpha 5 and strands beta 4-beta 6. Key residues in this cluster are Y320, which is crucial for the stabilization of the receptor-bound state, and F336, which stabilizes nucleotide-bound states. Destabilization of helix alpha 1, caused by rearrangement of this activation cluster, leads to the weakening of the interdomain interface and release of GDP.