A New Approach to Producing Functional Gα Subunits Yields the Activated and Deactivated Structures of Gα12/13 Proteins

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Abstract
The oncogenic G12/13 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 Gα12/13 subunits have been hindered by insufficient amounts of homogeneous, functional protein. Herein, we report that substitution of the N-terminal helix of Gαi1 for the corresponding region of Gα12 or Gα13 generated soluble chimeric subunits (Gαi/12 and Gαi/13) that could be purified in sufficient amounts for crystallographic studies. Each chimera bound guanine nucleotides, Gβγ subunits, and effector proteins and exhibited GAP responses to p115RhoGEF and leukemia-associated RhoGEF. Like their wild-type counterparts, Gαi/13, but not Gαi/12, stimulated the activity of p115RhoGEF. Crystal structures of the Gαi/12·GDP·AlF4- and Gαi/13·GDP complexes were determined using diffraction data extending to 2.9 and 2.0 Å, respectively. These structures reveal not only the native structural features of Gα12 and Gα13 subunits, which are expected to be important for their interactions with GPCRs and effectors such as Gα-regulated RhoGEFs, but also novel conformational changes that are likely coupled to GTP hydrolysis in the Gα12/13 class of heterotrimeric G proteins.