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A more recent version of this article appeared on November 11, 2005
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M505259200v1
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Papers In Press, published online ahead of print August 29, 2005
J. Biol. Chem, 10.1074/jbc.M505259200
Submitted on May 12, 2005
Revised on August 23, 2005
Accepted on August 29, 2005

Heterotrimeric G-protein alpha -subunit adopts a 'pre-activated' conformation when associated with beta gamma -subunits

Najmoutin G. Abdulaev, Tony Ngo, Cheng Zhang, Andy Dinh, Danielle M. Brabazon, Kevin D. Ridge, and John P. Marino

Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, Houston, TX 77030

Corresponding Author: Kevin.D.Ridge{at}uth.tmc.edu

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 (Galpha ) chimera (ChiT) associated with G-protein beta gamma -subunit (Gbeta gamma ) and activated receptor (R*) interactions. Here, we show that ChiT can be functionally reconstituted with Gbeta gamma as assessed by aluminum fluoride dependent changes in intrinsic tryptophan fluorescence, and light-activated rhodopsin catalyzed guanine nucleotide exchange. We further show that 15N-ChiT can be titrated with Gbeta gamma to form stable heterotrimers at NMR concentrations. To assess structural changes in ChiT upon heterotrimer formation, HSQC spectra of the 15N-ChiT reconstituted heterotrimer have been acquired and compared with spectra obtained for GDP/Mg2+-bound 15N-ChiT in the presence and absence of aluminum fluoride, and GTPgamma S/Mg2+-bound 15N-ChiT. As anticipated, Gbeta gamma association with 15N-ChiT results in 1HN, 15N chemical shift changes relative to the GDP/Mg2+-bound state. Strikingly, however, most 1HN, 15N chemical shift changes associated with heterotrimer formation are the same as those observed upon formation of the GDP•AlF4(super-}/Mg2+- and GTPgamma S/Mg2+-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 Galpha ('pre-activation') that may facilitate the interaction with R* and subsequent GDP/GTP exchange.


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