Download MendeleyA new approach to producing functional G alpha subunits yields the activated and deactivated structures of G alpha(12/13) proteins.
Kreutz, B., Yau, D.M., Nance, M.R., Tanabe, S., Tesmer, J.J., Kozasa, T.(2006) Biochemistry 45: 167-174
- PubMed: 16388592
- DOI: https://doi.org/10.1021/bi051729t
- Primary Citation of Related Structures:
1ZCA, 1ZCB - PubMed Abstract:
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.
Organizational Affiliation:
Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
