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

• DOI: 10.1021/bi051729t
• Publication Date: 2006-01-03T05:18:41Z
• Authors: Barry Kreutz; Douglas M. Yau; Mark R. Nance; Shihori Tanabe; John J. G. Tesmer; Tohru Kozasa
• Keywords: 0301 basic medicine; 0303 health sciences; Binding Sites; Leukemia; Chimera; Protein Conformation; GTP-Binding Protein beta Subunits; GTPase-Activating Proteins; Crystallography, X-Ray; GTP-Binding Protein alpha Subunits, G12-G13; Guanine Nucleotides; Receptors, G-Protein-Coupled; 03 medical and health sciences; GTP-Binding Protein gamma Subunits; Proto-Oncogene Proteins; Animals; Guanine Nucleotide Exchange Factors; Cells, Cultured; Rho Guanine Nucleotide Exchange Factors

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(i1) for the corresponding region of G alpha12 or G alpha13 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 betagamma 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(i/12) x GDP x AlF4(-) and G alpha(i/13) x GDP complexes were determined using diffraction data extending to 2.9 and 2.0 A, respectively. These structures reveal not only the native structural features of G alpha12 and G alpha13 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.