Crystal Structures of the G Protein G<sub>iα1</sub> Complexed with GDP and Mg<sup>2+</sup>:  A Crystallographic Titration Experiment

Coleman, David E.; Sprang, Stephen R.

doi:10.1021/bi9810306

Cited by 90 publications

(83 citation statements)

References 53 publications

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“…26 To gain insight into the cause of the red shift, we sought to compare structures of Ga i before and after activation, however, Ga i GDP is disordered in the Switch II region. 7,27 Therefore, structures of the closely related Ga i family member, Ga t were examined, since Ga t shares a high sequence and structural homology with Ga i . Comparison of the Switch II conformation in Ga t GDP and Ga t GDP-AlF 4 structures revealed an activation-dependent stacking of positively charged R204 (R208 in Ga i ) over the p electrons of W207 (W211 in Ga i ), reducing the distance between them by more than 2 Å [ Fig.…”

Section: Resultsmentioning

confidence: 99%

“…17 The Trp in Switch II (W211 in Ga i ) reports conformational changes upon activation. 20,21 The increase in emission intensity which accompanies the activationdependent movement of the Switch II region into a hydrophobic pocket [1][2][3][4][5][6][7] provides a convenient and reliable method to assess the functional integrity of purified proteins. 22,23 Ga i contains two other Trp residues, W258 in the GTPase domain and W131 in the helical domain.…”

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confidence: 99%

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Trp fluorescence reveals an activation‐dependent cation‐π interaction in the Switch II region of Gα_i proteins

et al. 2009

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Crystal structures of Ga i (and closely related family member Ga t ) reveal much of what we currently know about G protein structure, including changes which occur in Switch regions. Ga t exhibits a low rate of basal (uncatalyzed) nucleotide exchange and an ordered Switch II region in the GDP-bound state, unlike Ga i , which exhibits higher basal exchange and a disordered Switch II region in Ga i GDP structures. Using purified Ga i and Ga t , we examined the intrinsic tryptophan fluorescence of these proteins, which reports conformational changes associated with activation and deactivation of Ga proteins. In addition to the expected enhancement in tryptophan fluorescence intensity, activation of GaGDP proteins was accompanied by a modest but notable red shift in tryptophan emission maxima. We identified a cation-p interaction between tryptophan and arginine residues in the Switch II of Ga i family proteins that mediates the observed red shift in emission maxima. Furthermore, amino-terminal myristoylation of Ga 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 Ga proteins. These results reveal unique insights into conformational changes which occur upon activation and deactivation of G proteins in solution.

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Section: Resultsmentioning

confidence: 99%

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Trp fluorescence reveals an activation‐dependent cation‐π interaction in the Switch II region of Gα_i proteins

et al. 2009

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“…Fig. 4), we focused on the magnesium binding site of the G␣ subunit (26,27). Although magnesium effects on the activation of G␣ subunits have not been detected on G␣ i/o isolated from bovine brain (28), the fact that magnesium suppresses the guanine nucleotide exchange of small GTP-binding proteins (29) suggests the possibility of a similar mechanism for G␣ subunits.…”

Section: Effect Of Magnesium On the Activation Of A Casr Mutant With mentioning

confidence: 99%

Paradoxical Block of Parathormone Secretion Is Mediated by Increased Activity of Gα Subunits

Quitterer

Hoffmann

Freichel

et al. 2001

Journal of Biological Chemistry

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The paradox of blunted parathormone (PTH) secretion in patients with severe hypomagnesemia has been known for more than 20 years, but the underlying mechanism is not deciphered. We determined the effect of low magnesium on in vitro PTH release and on the signals triggered by activation of the calcium-sensing receptor (CaSR). Analogous to the in vivo situation, PTH release from dispersed parathyroid cells was suppressed under low magnesium. In parallel, the two major signaling pathways responsible for CaSR-triggered block of PTH secretion, the generation of inositol phosphates, and the inhibition of cAMP were enhanced. Desensitization or pertussis toxin-mediated inhibition of CaSR-stimulated signaling suppressed the effect of low magnesium, further confirming that magnesium acts within the axis CaSR-G-protein. However, the magnesium binding site responsible for inhibition of PTH secretion is not identical with the extracellular ion binding site of the CaSR, because the magnesium deficiency-dependent signal enhancement was not altered on CaSR receptor mutants with increased or decreased affinity for calcium and magnesium. By contrast, when the magnesium affinity of the G␣ subunit was decreased, CaSR activation was no longer affected by magnesium. Thus, the paradoxical block of PTH release under magnesium deficiency seems to be mediated through a novel mechanism involving an increase in the activity of G␣ subunits of heterotrimeric G-proteins. Parathormone (PTH)1 secretion from the parathyroid gland is suppressed by high extracellular calcium and magnesium (1). The calcium-sensing receptor (CaSR) is responsible for the calcium-dependent inhibition of PTH secretion (2). Direct binding of calcium or magnesium activates the CaSR (3). Activation of the CaSR triggers G␣ q /G␣ i -mediated signaling pathways (4). Several mutations have been identified with increased activation of this receptor (5, 6). CaSR mutants with increased affinity/potency for the agonist calcium and in part enhanced constitutive activity led to permanent inhibition of PTH secretion (7). Therefore, patients with activated CaSR mutants suffer from hypoparathyroidism. A similar phenotype of blunted PTH secretion is seen in patients with severe magnesium deficiency (8 -10). This finding is unexpected since the effects of high magnesium on parathyroid hormone secretion are similar to those of calcium, and therefore, low magnesium should be expected to result in increased PTH secretion. And indeed, in contrast to patients, rats respond to severe hypomagnesemia with increased secretion of PTH (11,12). It is known that hypomagnesemia reflects intracellular magnesium deficiency (9). Thus, the site of magnesium action has been assumed to lie intracellularly (9). However, causality between blunted PTH secretion and magnesium deficiency is not established, although the magnesium paradox has been known for more than 20 years (8). In search for the mechanism we investigated the relationship between magnesium deficiency, PTH secretion, and CaSR-mediated signaling...

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“…Crystallographic (4)(5)(6)(7)(8)(9), biochemical (10), and biophysical (11-13) studies have elucidated details of the conformational states of the Gα subunit during the GTPase cycle. The Gα subunit has two structural domains, a nucleotide-binding domain (the Ras-like domain) and a helical domain (the α-H domain) that partially occludes the bound nucleotide (Fig.…”

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confidence: 99%

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

Goricanec

Stehle

Egloff

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDPbound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape.H eterotrimeric G proteins are localized at the inner leaflet of the plasma membrane where they convey signals from cellsurface receptors to intracellular effectors (1). Heterotrimeric G proteins consist of two functional units, an α subunit (Gα) and a tightly associated βγ complex. The Gα subunit harbors the guanine nucleotide-binding site. In the inactive GDP-bound state, the Gα subunit is associated with the βγ complex. Exchange of GDP for GTP on the Gα subunit, triggered by interaction with the agonist-bound G-protein-coupled receptor (GPCR), results in a conformational change leading to GDP release and ultimately to GTP binding and subunit dissociation. The complexity of the mechanism by which a GPCR activates the Gα subunit based on available crystal structures has been discussed recently (2, 3). Both the Gα subunit and the βγ subunit interact with downstream effectors and regulate their activity. The intrinsic GTP hydrolysis of the Gα subunit returns the protein to the GDP-bound state, thereby increasing its affinity for the Gβγ subunit, and the subunits reassociate (Fig. 1A), ready for interaction with the agonist-bound GPCR. Throughout this cycle, the Gα subunit is engaged in specific interactions with the GPCR and/or the βγ subunit that stabilize the flexible parts of the protein, e.g., its switch regions. Only the GTP-bound form is stable enough to mediate downstream signaling.Crystallographic (4-9), biochemical (10), and biophysical (11-13) studies have elucidated details of the conformational states of the Gα subunit during the GTPase cycle. The Gα subunit has two structural domains, a nucleotide-binding domain (the Ras-like domain) and a helical domain (the α-H domain) that partially occludes the bound nucleotide (Fig. 1A). Because of this steric considerati...

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Crystal Structures of the G Protein G_iα1 Complexed with GDP and Mg²⁺: A Crystallographic Titration Experiment

Cited by 90 publications

References 53 publications

Trp fluorescence reveals an activation‐dependent cation‐π interaction in the Switch II region of Gα_i proteins

Trp fluorescence reveals an activation‐dependent cation‐π interaction in the Switch II region of Gα_i proteins

Paradoxical Block of Parathormone Secretion Is Mediated by Increased Activity of Gα Subunits

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

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Crystal Structures of the G Protein Giα1 Complexed with GDP and Mg2+: A Crystallographic Titration Experiment

Cited by 90 publications

References 53 publications

Trp fluorescence reveals an activation‐dependent cation‐π interaction in the Switch II region of Gαi proteins

Trp fluorescence reveals an activation‐dependent cation‐π interaction in the Switch II region of Gαi proteins

Paradoxical Block of Parathormone Secretion Is Mediated by Increased Activity of Gα Subunits

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

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Crystal Structures of the G Protein G_iα1 Complexed with GDP and Mg²⁺: A Crystallographic Titration Experiment

Trp fluorescence reveals an activation‐dependent cation‐π interaction in the Switch II region of Gα_i proteins

Trp fluorescence reveals an activation‐dependent cation‐π interaction in the Switch II region of Gα_i proteins