title: Heterotrimeric G-protein alpha-subunit adopts a "preactivated" conformation when associated with betagamma-subunits.
abstract: Activation of a heterotrimeric G-protein by an agonist-stimulated G-protein-coupled receptor requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide binding pocket of the G-protein. To probe the molecular basis of this signaling process, we are applying high resolution NMR to track structural changes in an isotope-labeled, full-length G-protein alpha-subunit (G(alpha)) chimera (ChiT) associated with G-protein betagamma-subunit (G(betagamma)) and activated receptor (R(*)) interactions. Here, we show that ChiT can be functionally reconstituted with G(betagamma) as assessed by aluminum fluoride-dependent changes in intrinsic tryptophan fluorescence and light-activated rhodopsin-catalyzed guanine nucleotide exchange. We further show that (15)N-ChiT can be titrated with G(betagamma) to form stable heterotrimers at NMR concentrations. To assess structural changes in ChiT upon heterotrimer formation, HSQC spectra of the (15)N-ChiT-reconstituted heterotrimer have been acquired and compared with spectra obtained for GDP/Mg(2+)-bound (15)N-ChiT in the presence and absence of aluminum fluoride and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS)/Mg(2+)-bound (15)N-ChiT. As anticipated, G(betagamma) association with (15)N-ChiT results in (1)HN, (15)N chemical shift changes relative to the GDP/Mg(2+)-bound state. Strikingly, however, most (1)HN, (15)N chemical shift changes associated with heterotrimer formation are the same as those observed upon formation of the GDP.AlF(4)(-)/Mg(2+)- and GTPgammaS/Mg(2+)-bound states. Based on these comparative analyses, assembly of the heterotrimer appears to induce structural changes in the switch II and carboxyl-terminal regions of G(alpha) ("preactivation") that may facilitate the interaction with R(*) and subsequent GDP/GTP exchange.
authors.affiliation: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute and National Institute of Standards and Technology, Rockville, 20850, USA.
authors.forename: Najmoutin G
authors.initials: NG
authors.lastname: Abdulaev
authors.forename: Tony
authors.initials: T
authors.lastname: Ngo
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhang
authors.forename: Andy
authors.initials: A
authors.lastname: Dinh
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Brabazon
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Ridge
authors.forename: John P
authors.initials: JP
authors.lastname: Marino
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry* / Q000235:genetics / 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*; D015870:Gene Expression; D008274:Magnesium; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D008274:Magnesium