A new approach to producing functional G alpha subunits yields the activated and deactivated structures of G alpha(12/13) 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(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.
Kreutz
Barry
B
Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
Yau
Douglas M
DM
Nance
Mark R
MR
Tanabe
Shihori
S
Tesmer
John J G
JJ
Kozasa
Tohru
T
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D002678:Chimera / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D006150:Guanine Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007938:Leukemia / Q000378:metabolism; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors
C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides; D011518:Proto-Oncogene Proteins; D043562:Receptors, G-Protein-Coupled; D064067:Rho Guanine Nucleotide Exchange Factors; D044365:GTP-Binding Protein alpha Subunits, G12-G13