title: The receptor-bound "empty pocket" state of the heterotrimeric G-protein alpha-subunit is conformationally dynamic.
abstract: Heterotrimeric G-protein activation by a G-protein-coupled receptor (GPCR) requires the propagation of structural signals from the receptor-interacting surfaces to the guanine nucleotide-binding pocket. To probe conformational changes in the G-protein alpha-subunit (G(alpha)) associated with activated GPCR (R*) interactions and guanine nucleotide exchange, high-resolution solution NMR methods are being applied in studying signaling of the G-protein, transducin, by light-activated rhodopsin. Using these methods, we recently demonstrated that an isotope-labeled G(alpha) reconstituted heterotrimer forms functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin and a soluble mimic of R*, both of which trigger guanine nucleotide exchange [Ridge, K. D., et al. (2006) J. Biol. Chem. 281, 7635-7648]. Here, it is shown that both light-activated rhodopsin and the soluble mimic of R form trapped intermediate complexes with a GDP-released "empty pocket" state of the heterotrimer in the absence of GTP (or GTPgammaS). In contrast to guanine nucleotide-bound forms of G(alpha), the NMR spectra of the GDP-released, R-bound empty pocket state of G(alpha) display severe line broadening suggestive of a dynamic intermediate state. Interestingly, the conformation of a GDP-depleted, Mg(2+)-bound state of G(alpha) generated in a manner independent of R* does not exhibit a similar degree of line broadening but rather appears structurally similar to the GDP/Mg(2+)-bound form of the protein. Taken together, these results suggest that R*-mediated changes in the receptor-interacting regions of G(alpha), and not the absence of bound guanine nucleotide, are the predominant factors which dictate G(alpha) conformation and dynamics in this R*-bound state of the heterotrimer.
authors.affiliation: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute and National Institute of Standards and Technology, Rockville, Maryland 20850, USA.
authors.forename: Najmoutin G
authors.initials: NG
authors.lastname: Abdulaev
authors.forename: Tony
authors.initials: T
authors.lastname: Ngo
authors.forename: Eva
authors.initials: E
authors.lastname: Ramon
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Brabazon
authors.forename: John P
authors.initials: JP
authors.lastname: Marino
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Ridge
mesh_terms: D000818:Animals; D002417:Cattle; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry* / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D008027:Light; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding / Q000528:radiation effects; D011487:Protein Conformation / Q000528:radiation effects; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism; D015293:Transducin / Q000737:chemistry / Q000378:metabolism
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D006150:Guanine Nucleotides; D043562:Receptors, G-Protein-Coupled; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D020962:Heterotrimeric GTP-Binding Proteins; D015293:Transducin