title: Functional reconstitution of an atypical G protein heterotrimer and regulator of G protein signaling protein (RGS1) from Arabidopsis thaliana.
abstract: It has long been known that animal heterotrimeric Gαβγ proteins are activated by cell-surface receptors that promote GTP binding to the Gα subunit and dissociation of the heterotrimer. In contrast, the Gα protein from Arabidopsis thaliana (AtGPA1) can activate itself without a receptor or other exchange factor. It is unknown how AtGPA1 is regulated by Gβγ and the RGS (regulator of G protein signaling) protein AtRGS1, which is comprised of an RGS domain fused to a receptor-like domain. To better understand the cycle of G protein activation and inactivation in plants, we purified and reconstituted AtGPA1, full-length AtRGS1, and two putative Gβγ dimers. We show that the Arabidopsis Gα protein binds to its cognate Gβγ dimer directly and in a nucleotide-dependent manner. Although animal Gβγ dimers inhibit GTP binding to the Gα subunit, AtGPA1 retains fast activation in the presence of its cognate Gβγ dimer. We show further that the full-length AtRGS1 protein accelerates GTP hydrolysis and thereby counteracts the fast nucleotide exchange rate of AtGPA1. Finally, we show that AtGPA1 is less stable in complex with GDP than in complex with GTP or the Gβγ dimer. Molecular dynamics simulations and biophysical studies reveal that altered stability is likely due to increased dynamic motion in the N-terminal α-helix and Switch II of AtGPA1. Thus, despite profound differences in the mechanisms of activation, the Arabidopsis G protein is readily inactivated by its cognate RGS protein and forms a stable, GDP-bound, heterotrimeric complex similar to that found in animals.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Janice C
authors.initials: JC
authors.lastname: Jones
authors.forename: Brenda R S
authors.initials: BR
authors.lastname: Temple
authors.forename: Alan M
authors.initials: AM
authors.lastname: Jones
authors.forename: Henrik G
authors.initials: HG
authors.lastname: Dohlman
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; C429765:GPA1 protein, Arabidopsis; D044385:GTP-Binding Protein alpha Subunits; D020710:RGS Proteins; C477755:RGS1 protein, Arabidopsis; D006160:Guanosine Triphosphate