Conformational Changes Associated with Receptor-stimulated Guanine Nucleotide Exchange in a Heterotrimeric G-protein alpha -Subunit: NMR Analysis of GTP gamma S-Bound States

• Published in: The Journal of biological chemistry 2006-03, Vol.281 (11), p.7635-7648
• Main Authors: Ridge, Kevin D, Abdulaev, Najmoutin G, Zhang, Cheng, Ngo, Tony, Brabazon, Danielle M, Marino, John P
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M509851200

Solution NMR studies of a super(15)N-labeled G-protein alpha -subunit (G sub( alpha )) chimera ( super(15)N-ChiT)-reconstituted heterotrimer have shown previously that G-protein beta gamma -subunit (G sub( beta gamma )) association induces a "pre-activated" conformation that likely facilitates interaction with the agonist-activated form of a G-protein-coupled receptor (R super(*)) 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 super(15)N-ChiT-reconstituted heterotrimer can form functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin (R super(*)), as well as a soluble mimic of R super(*). NMR methods were used to track R super(*)-triggered guanine nucleotide exchange and release of guanosine 5'-O-3-thiotriphosphate (GTP gamma S)/Mg super(2+)-bound ChiT. A heteronuclear single quantum correlation (HSQC) spectrum of R super(*)-generated GTP gamma S/Mg super(2+)-bound ChiT revealed super(1)HN, super(15)N chemical shift changes relative to GDP/Mg super(2+)-bound ChiT that were similar, but not identical, to those observed for the [Formula: see text]-bound state. Line widths observed for R super(*)-generated GTP gamma S/Mg super(2+)-bound super(15)N-ChiT, however, indicated that it is more conformationally dynamic relative to the GDP/Mg super(2+)- and [Formula: see text]-bound states. The increased dynamics appeared to be correlated with G sub( beta gamma ) and R super(*) interactions because they are not observed for GTP gamma S/Mg super(2+)-bound ChiT generated independently of R super(*). In contrast to R super(*), 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 GTP gamma S/Mg super(2+)-exchanged heterotrimer. The HSQC spectrum of super(15)N-ChiT in this complex displays a unique chemical shift pattern that nonetheless shares similarities with the heterotrimer and GTP gamma S/Mg super(2+)-bound ChiT. Overall, these results demonstrated that R super(*)-induced changes in G sub( alpha ) can be followed by NMR and that guanine nucleotide exchange can be uncoupled from heterotrimer dissociation.