ABSTRACT
Crystal structures of Galpha(i) (and closely related family member Galpha(t)) reveal much of what we currently know about G protein structure, including changes which occur in Switch regions. Galpha(t) exhibits a low rate of basal (uncatalyzed) nucleotide exchange and an ordered Switch II region in the GDP-bound state, unlike Galpha(i), which exhibits higher basal exchange and a disordered Switch II region in Galpha(i)GDP structures. Using purified Galpha(i) and Galpha(t), we examined the intrinsic tryptophan fluorescence of these proteins, which reports conformational changes associated with activation and deactivation of Galpha proteins. In addition to the expected enhancement in tryptophan fluorescence intensity, activation of GalphaGDP proteins was accompanied by a modest but notable red shift in tryptophan emission maxima. We identified a cation-pi interaction between tryptophan and arginine residues in the Switch II of Galpha(i) family proteins that mediates the observed red shift in emission maxima. Furthermore, amino-terminal myristoylation of Galpha(i) resulted in a less polar environment for tryptophan residues in the GTPase domain, consistent with an interaction between the myristoylated amino terminus and the GTPase domain of Galpha proteins. These results reveal unique insights into conformational changes which occur upon activation and deactivation of G proteins in solution.
Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Models, Molecular , Protein Structure, Tertiary/genetics , Tryptophan/metabolism , Arginine/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Guanosine Diphosphate/metabolism , Kinetics , Myristic Acid/metabolism , Protein Conformation , Spectrometry, Fluorescence/methodsABSTRACT
Heterotrimeric G proteins (Galphabetagamma) transmit signals from activated G protein-coupled receptors (GPCRs) to downstream effectors through a guanine nucleotide signaling cycle. Numerous studies indicate that the carboxy-terminal alpha5 helix of Galpha subunits participates in Galpha-receptor binding, and previous EPR studies suggest this receptor-mediated interaction induces a rotation and translation of the alpha5 helix of the Galpha subunit [Oldham, W. M., et al. (2006) Nat. Struct. Mol. Biol. 13, 772-777]. On the basis of this result, an engineered disulfide bond was designed to constrain the alpha5 helix of Galpha(i1) into its EPR-measured receptor-associated conformation through the introduction of cysteines at position 56 in the alpha1 helix and position 333 in the alpha5 helix (I56C/Q333C Galpha(i1)). A functional mimetic of the EPR-measured alpha5 helix dipole movement upon receptor association was additionally created by introduction of a positive charge at the amino terminus of this helix, D328R Galpha(i1). Both proteins exhibit a dramatically elevated level of basal nucleotide exchange. The 2.9 A resolution crystal structure of I56C/Q333C Galpha(i1) in complex with GDP-AlF(4)(-) reveals the shift of the alpha5 helix toward the guanine nucleotide binding site that is anticipated by EPR measurements. The structure of the I56C/Q333C Galpha(i1) subunit further revealed altered positions for the switch regions and throughout the Galpha(i1) subunit, accompanied by significantly elevated crystallographic temperature factors. Combined with previous evidence in the literature, the structural analysis supports the critical role of electrostatics of the alpha5 helix dipole and overall conformational variability during nucleotide release.
Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , Guanosine Diphosphate/metabolism , Allosteric Site , Binding Sites , Crystallography, X-Ray , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Guanosine Diphosphate/chemistry , Kinetics , Models, Molecular , Protein Conformation , Protein Subunits , Spectrometry, FluorescenceABSTRACT
G protein-coupled receptors (GPCRs) catalyze nucleotide release in heterotrimeric G proteins, the slow step in G protein activation. G i/o family proteins are permanently, cotranslationally myristoylated at the extreme amino terminus. While myristoylation of the amino terminus has long been known to aid in anchoring G i proteins to the membrane, the role of myristoylation with regard to interaction with activated receptors is not known. Previous studies have characterized activation-dependent changes in the amino terminus of Galpha proteins in solution [Medkova, M. (2002) Biochemistry 41, 9963-9972; Preininger, A. M. (2003) Biochemistry 42, 7931-7941], but changes in the environment of specific residues within the Galpha i1 amino terminus during receptor-mediated G i activation have not been reported. Using site-specific fluorescence labeling of individual residues along a stretch of the Galpha il amino terminus, we found specific changes in the environment of these residues upon interaction with the activated receptor and following GTPgammaS binding. These changes map to a distinct surface of the amino-terminal helix opposite the Gbetagamma binding interface. The receptor-dependent fluorescence changes are consistent with a myristoylated amino terminus in the proximity of the membrane and/or receptor. Myristoylation affects both the rate and intensity of receptor activation-dependent changes detected at several residues along the amino terminus (with no significant effect on the rate of receptor-mediated GTPgammaS binding). This work demonstrates that the myristoylated amino terminus of Galpha il proteins undergoes receptor-mediated changes during the dynamic process of G protein signaling.
