A new approach to producing functional Gα subunits yields the activated and deactivated structures of Gα12/13 proteins

The oncogenic G(12/13) subfamily of heterotrimeric G proteins transduces extracellular signals that regulate the actin cytoskeleton, cell cycle progression, and gene transcription. Previously, structural analyses of fully functional G alpha(12/13) subunits have been hindered by insufficient amounts of homogeneous, functional protein. Herein, we report that substitution of the N-terminal helix of G alpha(il) for the corresponding region of G alpha(12) or G alpha(13) generated soluble chimeric subunits (G alpha(i/12) and G(alpha(i/13)) that could be purified in sufficient amounts for crystallographic studies. Each chimera bound guanine nucleotides, G beta gamma Subunits, and effector proteins and exhibited GAP responses to p115RhoGEF and leukemia-associated RhoGEF. Like their wild-type counterparts, G alpha(i/13), but not G alpha(i/12), stimulated the activity of p115RhoGEF. Crystal structures of the G alpha(circle)(i/12)GDP(circle)AlF(4)(-) and G alpha(i/13)-GDP complexes were determined using diffraction data extending to 2.9 and 2.0 angstrom, respectively. These structures reveal not only the native structural features of G alpha(12) and G alpha(13) subunits, which are expected to be important for their interactions with GPCRs and effectors such as G alpha-regulated RhoGEFs, but also novel conformational changes that are likely coupled to GTP hydrolysis in the G alpha(12/13) class of heterotrimeric G proteins.