Conformational changes associated with receptor-stimulated guanine nucleotide exchange in a heterotrimeric G-protein alpha-subunit: NMR analysis of GTPgammaS-bound states.
Solution NMR studies of a (15)N-labeled G-protein alpha-subunit (G(alpha)) chimera ((15)N-ChiT)-reconstituted heterotrimer have shown previously that G-protein betagamma-subunit (G(betagamma)) association induces a "pre-activated" conformation that likely facilitates interaction with the agonist-activated form of a G-protein-coupled receptor (R*) and guanine nucleotide exchange (Abdulaev, N. G., Ngo, T., Zhang, C., Dinh, A., Brabazon, D. M., Ridge, K. D., and Marino, J. P. (2005) J. Biol. Chem. 280, 38071-38080). Here we demonstrated that the (15)N-ChiT-reconstituted heterotrimer can form functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin (R*), as well as a soluble mimic of R*. NMR methods were used to track R*-triggered guanine nucleotide exchange and release of guanosine 5'-O-3-thiotriphosphate (GTPgammaS)/Mg(2+)-bound ChiT. A heteronuclear single quantum correlation (HSQC) spectrum of R*-generated GTPgammaS/Mg(2+)-bound ChiT revealed (1)HN, (15)N chemical shift changes relative to GDP/Mg(2+)-bound ChiT that were similar, but not identical, to those observed for the GDP.AlF(4)(-)/Mg(2+)-bound state. Line widths observed for R*-generated GTPgammaS/Mg(2+)-bound (15)N-ChiT, however, indicated that it is more conformationally dynamic relative to the GDP/Mg(2+)- and GDP.AlF(4)(-)/Mg(2+)-bound states. The increased dynamics appeared to be correlated with G(betagamma) and R* interactions because they are not observed for GTPgammaS/Mg(2+)-bound ChiT generated independently of R*. In contrast to R*, a soluble mimic that does not catalytically interact with G-protein (Abdulaev, N. G., Ngo, T., Chen, R., Lu, Z., and Ridge, K. D. (2000) J. Biol. Chem. 275, 39354-39363) is found to form a stable complex with the GTPgammaS/Mg(2+)-exchanged heterotrimer. The HSQC spectrum of (15)N-ChiT in this complex displays a unique chemical shift pattern that nonetheless shares similarities with the heterotrimer and GTPgammaS/Mg(2+)-bound ChiT. Overall, these results demonstrated that R*-induced changes in G(alpha) can be followed by NMR and that guanine nucleotide exchange can be uncoupled from heterotrimer dissociation.
Ridge
Kevin D
KD
Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, TX 77030, USA. Kevin.D.Ridge@uth.tmc.edu
Abdulaev
Najmoutin G
NG
Zhang
Cheng
C
Ngo
Tony
T
Brabazon
Danielle M
DM
Marino
John P
JP
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology; D019281:Dimerization; D006147:Guanine / Q000737:chemistry; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry*; D047468:Immunoprecipitation; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry; D017299:Rod Opsins / Q000737:chemistry; D020860:Subtilisin / Q000737:chemistry; D013997:Time Factors
D003902:Detergents; D020662:Guanine Nucleotide Exchange Factors; D011993:Recombinant Fusion Proteins; D017299:Rod Opsins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006147:Guanine; C013110:metarhodopsins; D012243:Rhodopsin; D020860:Subtilisin; D008274:Magnesium